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  • 机译 缩回:骨间充质干细胞条件培养基通过Notch通路激活来调节神经干细胞的分化,作者:Li H-M,Tong Y,Xia X,Huang J,Song P-W,Zhang R-J,Shen C-L。单元重编程。 [Epub提前发布; 2018年12月27日] DOI:10.1089 / cell.2018.0042
    • 作者:
    • 刊名:Cellular Reprogramming
    • 2019年第1期
    摘要:
  • 机译 通过额外的辅助细胞质提高克隆牛胚胎的体外发育能力和重编程效率
    摘要:Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker® Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.
  • 机译 利用人外周血单核细胞制备诱导性多能干细胞
    摘要:Since induced pluripotent stem (iPS) cells have been established, in recent years, clinical transplantation of cells differentiated from iPS cells derived from human skin fibroblasts is been in progress. On the contrary, monocytes have complete genome information without damage and gene recombination, they are contained in the peripheral blood by ∼3%–8% and differentiate into dendritic cells that are the type of control tower for immune cells. However, generation of monocyte-derived iPS cells has only been successful when special persistent Sendai virus vectors have been used. Therefore, in this study, as a preculture method for monocytes, a culture method for maintaining activity without using any cytokine was established, and using a commercially available vector without genetic toxicity without damaging the chromosome of the cell, iPS cells derived from monocytes were successfully produced. This cell has the ability to differentiate into three germ layers, and when compared with commercially available iPS cells, there was no significant difference between self-renewal and gene expression in the three germ layers. In future, we will compare the differentiation induction of monocyte-derived iPS cells with dendritic cells and investigate the production of dendritic cells that can cope with various antigens.
  • 机译 从马诱导多能干细胞产生功能性心肌细胞
    摘要:Induced pluripotent stem cells (iPSCs) have revolutionized human biomedicine through their use in disease modeling and therapy. In comparison, little progress has been made toward the application of iPSCs in veterinary species. In that regard, skeletal myocytes from iPSCs would have great potential for understanding muscle function and disease in the equine athlete. In this study, we generated skeletal myotubes by transducing equine iPSC-derived mesenchymal derivatives with an inducible lentiviral vector coding for the human sequence of the myogenic factor, MyoD. Myosin heavy chain-positive myotubes generated from two different iPSC lines were compared to myotubes from adult equine skeletal muscle progenitor cells (MPCs). iPSC myotubes had a smaller mean area than MPC myotubes (≤2-fold). In addition, quantitative polymerase chain reaction analyses showed that iPSC myotubes expressed MYH2 and MYH3 isoforms (at similar or lower levels than MPC myotubes), but they did not express the mature muscle isoform, MYH1. Compared to MPC myotubes, iPSC myotubes expressed reduced levels of the myogenic factors, MYOD1 and MYF6, but did not express MYF5. Finally, iPSC myotubes responded to KCl-induced membrane depolarization by releasing calcium and did so in a manner similar to MPC myotubes. In conclusion, this is the first study to report the generation of functional myocytes from equine iPSCs.
  • 机译 猪胎儿成纤维细胞改变基因表达和线粒体以补偿培养过程中的低氧应激
    摘要:The Warburg effect is characterized by decreased mitochondrial oxidative phosphorylation and increased glycolytic flux in adequate oxygen. The preimplantation embryo has been described to have characteristics of the Warburg effect, including similar changes in gene expression and mitochondria, which are more rudimentary in appearance. We hypothesized hypoxia would facilitate anaerobic glycolysis in fibroblasts thereby promoting gene expression and media metabolite production reflecting the Warburg effect hallmarks in early embryos. Additionally, we speculated that hypoxia would induce a rudimentary small mitochondrial phenotype observed in several cell types evidenced to demonstrate the Warburg effect. While many have examined the role hypoxia plays in pathological conditions, few studies have investigated changes in primary cells which could be used in somatic cell nuclear transfer. We found that cells grown in 1.25% O2 had normal cell viability and more, but smaller mitochondria. Several hypoxia-inducible genes were identified, including seven genes for glycolytic enzymes. In conditioned media from hypoxic cells, the quantities of gluconolactone, cytosine, and uric acid were decreased indicating higher consumption than control cells. These results indicate that fibroblasts alter gene expression and mitochondria to compensate for hypoxic stress and maintain viability. Furthermore, the metabolic changes observed, making them more similar to preimplantation embryos, could be facilitating nuclear reprogramming making these cells more amendable to future use in somatic cell nuclear transfer.
  • 机译 串行培养对于金叙利亚仓鼠单性生殖胚胎的体外发育至关重要
    摘要:Unlike oocytes of many other mammalian species, parthenogenetically activated hamster oocytes have not been reported to develop beyond the two-cell stage. This study investigated the in vitro development into blastocysts of parthenogenetic embryos of Golden Syrian hamsters. We observed that hamster oocytes could easily be artificially activated (AA) by treatment with ionomycin plus 6-dimethylaminopurine + cycloheximide + cytochalasin B as assessed by embryo cleavage in HECM-9 (63.15%) or HECM-10 (63.82%). None of the cleaved embryos developed beyond the two-cell stage when cultured in either of the two media. However, some of the embryos overcame the two-cell block and developed to the blastocyst stage (26.45%) when they were first cultured in HECM-10 for 24 hours and then in HECM-9 (serial culture media HECM-10-9) for 72 hours. Blastocyst development was further significantly (66.2%) improved when embryos were cultured in HECM-10 supplemented with ethylenediaminetetraacetic acid for 24 hours, then in HECM-9 supplemented with glucose for 72 hours (serial culture media HECM-11a-b). Hamster oocytes activated with ionomycin, ethanol, or a combination of the two treatments would develop to the blastocyst stage in serial culture media HECM-11a-b, whereas none of the spontaneously activated oocytes cleaved (0% vs. 86.93%, p < 0.05). DNA and microtubule configurations of spontaneously activated and AA oocytes were assessed by immunocytochemical staining and fluorescence microscopy. The results indicate that serial culture and the method of activation are critical for overcoming the in vitro developmental block of hamster parthenogenetic embryos. This study is the first to report blastocyst development from parthenogenetically activated hamster oocytes.
  • 机译 在小鼠胚胎成纤维细胞向心肌细胞转分化中增加Oct4和抑制p53的作用的互惠性
    摘要:p53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53+/+), p53 heterozygous mutant (p53+/−), and p53 homozygous mutant (p53−/−) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53+/− and p53−/− MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53+/− and p53−/− MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53+/+ and p53+/− MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53+/− MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.
  • 机译 人诱导的多能干细胞的麻疹病毒持续感染
    摘要:In this study, we found that the measles virus (MV) can infect human-induced pluripotent stem cells (hiPSCs). Wild-type MV strains generally use human signaling lymphocyte activation molecule (SLAM; CD150) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both CD150 and CD46 as receptors. It is not yet known how early in the embryonal differentiation stages these receptors are expressed. We established two hiPSCs (BGU-iPSCs and EMF-iPSCs) which express CD46 and CD150. Both cell types can be infected by MV to form persistent, noncytopathic cell lines that release infectious MV particles. Following MV persistent infection, BGU-iPSCs and EMF-iPSCs remain pluripotent and can differentiate in vitro into the three germ layers. This includes cells expressing the neuronal differentiation markers: NF68 and miRNA-124. Since the MV does not integrate into the cell's genome, it can be utilized as a vehicle to systematically introduce genes into iPSC, to dissect and to define factors regulating lineage differentiation.
  • 机译 药理学重编程旨在在猪胎儿成纤维细胞中引起Warburg效应而改变基因表达和条件培养基中代谢物的数量,而不会增加细胞增殖
    摘要:The Warburg effect is a metabolic phenomenon characterized by increased glycolytic activity, decreased mitochondrial oxidative phosphorylation, and the production of lactate. This metabolic phenotype is characterized in rapidly proliferative cell types such as cancerous cells and embryonic stem cells. We hypothesized that a Warburg-like metabolism could be achieved in other cell types by treatment with pharmacological agents, which might, in turn, facilitate nuclear reprogramming. The aim of this study was to treat fibroblasts with CPI-613 and PS48 to induce a Warburg-like metabolic state. We demonstrate that treatment with both drugs altered the expression of 69 genes and changed the level of 21 metabolites in conditioned culture media, but did not induce higher proliferation compared to the control treatment. These results support a role for the reverse Warburg effect, whereby cancer cells induce cancer-associated fibroblast cells in the surrounding stroma to exhibit the metabolically characterized Warburg effect. Cancer-associated fibroblasts then produce and secrete metabolites such as pyruvate to supply the cancerous cells, thereby supporting tumor growth and metastasis. While anticipating an increase in the production of lactate and increased cellular proliferation, both hallmarks of the Warburg effect, we instead observed increased secretion of pyruvate without changes in proliferation.
  • 机译 克隆牛的无尾性不是遗传传播的
    摘要:Somatic cell nuclear transfer (SCNT), commonly referred to as cloning, results in the generation of offspring that, except for mitochondrial DNA, are genetically identical to the nuclear donor. We previously used a genetically modified bovine cell line as the donor for SCNT and obtained a calf, named Daisy, that was born without a tail. To determine whether the missing tail was a result of the genetic modification, we performed recloning experiments by using either cells from a sacrificed pregnancy of a second clone (Daisy's “twin” clone) or cells from tailless Daisy as donors for SCNT. Cloned fetuses from aborted pregnancies and a cloned live calf that died shortly after birth were examined and confirmed to all possess tails. Hence, the observed phenotype of Daisy's lacking tail is not due to the introduced transgene or a mutation present in the cell that was used for her production. Rather, the missing tail has most likely arisen from an epigenetic reprogramming error during development.
  • 机译 HSP90抑制和细胞应激在造血分化中引起表型可塑性。
    摘要:Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%–90% of the cells die when placed in medium where the major growth factor is granulocyte–macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.
  • 机译 YY1是SOX2和OCT4蛋白质转录后稳定性所需的
    摘要:Yinyang1 (YY1) participates in protein-DNA, protein-RNA, and protein–protein interactions and regulates developmental processes and disease mechanisms. YY1 interactions regulate a range of important biological functions, including oocyte maturation, epithelial to mesenchymal transition, and vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that YY1 is required for inner cell mass (ICM) lineage commitment during preimplantation development. In this study, we document gene expression patterns and protein localization of key transcription factors in Yy1 global, tissue-specific, and dsRNA-mediated knockout/down embryos. YY1 protein was found in cells of preimplantation and peri-implantation embryos, and adult tissues where two isoforms are observed. In the absence of YY1, OCT4 and SOX2 protein were lost in the ICM during preimplantation and naive neuroectoderm during gastrulation stages, yet no difference in Oct4 or Sox2 mRNA levels was observed. The loss of OCT4 and SOX2 protein occurred specifically in cells that normally express both OCT4 and SOX2 protein. These observations support a role for YY1 meditating and/or regulating the interaction of OCT4 and SOX2 at a posttranscriptional level. Our results suggest that distinct mechanisms of YY1-mediated molecular regulation are present in the early embryo, and may offer insight to promote lineage commitment in in vitro cell lines.
  • 机译 激活素A或烟酰胺与胰腺转录因子PDX1的组合支持人羊膜上皮细胞向胰腺谱系分化
    摘要:The differentiation of multipotent stem cells toward a pancreatic lineage provides us with an alternative cell-based therapeutic approach to type 1 diabetes and enables us to study pancreas development. The current study aims to study the effect of growth factors such as activin A or nicotinamide, alone and in combinations with the transcription factor, PDX1 (pancreatic and duodenal homeobox-1), on human amnion epithelial cells (hAECs) toward a pancreatic lineage. Ectopic expression of Pdx1 followed by treatment of hAECs with nicotinamide for 4 days resulted in strong induction of pancreatic endoderm and pancreatic progenitor genes, including NKX6.1 and NEUROD1. Pancreatic lineage cells expressing PDX1, SOX17, and RFX6 are derived from Pdx1-transduced hAECs treated with activin A or nicotinamide, but not cells treated with activin A or nicotinamide alone. Our study provides a novel culture protocol for generating pancreas-committed cells from hAECs and reveals an interplay between Pdx1 and activin A/nicotinamide signaling in early pancreatic fate determination.
  • 机译 猪诱导多能干细胞衍生的神经玫瑰花丛并行人类分化成感觉神经亚型。
    摘要:The pig is the large animal model of choice for study of nerve regeneration and wound repair. Availability of porcine sensory neural cells would conceptually allow for analogous cell-based peripheral nerve regeneration in porcine injuries of similar severity and size to those found in humans. After recently reporting that porcine (or pig) induced pluripotent stem cells (piPSCs) differentiate into neural rosette (NR) structures similar to human NRs, here we demonstrate that pig NR cells could differentiate into neural crest cells and other peripheral nervous system-relevant cell types. Treatment with either bone morphogenetic protein 4 or fetal bovine serum led to differentiation into BRN3A-positive sensory cells and increased expression of sensory neuron TRK receptor gene family: TRKA, TRKB, and TRKC. Porcine sensory neural cells would allow determination of parallels between human and porcine cells in response to noxious stimuli, analgesics, and reparative mechanisms. In vitro differentiation of pig sensory neurons provides a novel model system for neural cell subtype specification and would provide a novel platform for the study of regenerative therapeutics by elucidating the requirements for innervation following injury and axonal survival.
  • 机译 转化生长因子β信号通路对鸡胚胎干细胞向雄性生殖细胞分化的影响
    摘要:The objectives of the present study were to screen for key gene and signaling pathways involved in the production of male germ cells in poultry and to investigate the effects of the transforming growth factor beta (TGF-β) signaling pathway on the differentiation of chicken embryonic stem cells (ESCs) into male germ cells. The ESCs, primordial germ cells, and spermatogonial stem cells (SSCs) were sorted using flow cytometry for RNA sequencing (RNA-seq) technology. Male chicken ESCs were induced using 40 ng/mL of bone morphogenetic protein 4 (BMP4). The effects of the TGF-β signaling pathway on the production of chicken SSCs were confirmed by morphology, quantitative real-time polymerase chain reaction, and immunocytochemistry. One hundred seventy-three key genes relevant to development, differentiation, and metabolism and 20 signaling pathways involved in cell reproduction, differentiation, and signal transduction were identified by RNA-seq. The germ cells formed agglomerates and increased in number 14 days after induction by BMP4. During the induction process, the ESCs, Nanog, and Sox2 marker gene expression levels decreased, whereas expression of the germ cell-specific genes Stra8, Dazl, integrin-α6, and c-kit increased. The results indicated that the TGF-β signaling pathway participated in the differentiation of chicken ESCs into male germ cells.
  • 机译 非整合型仙台病毒从糖尿病足溃疡成纤维细胞诱导多能干细胞的生成。
    摘要:Diabetic foot ulcers (DFUs) are nonhealing chronic wounds that are a serious complication of diabetes. Since induced pluripotent stem cells (iPSCs) may offer a potent source of autologous cells to heal these wounds, we studied if repair-deficient fibroblasts, derived from DFU patients and age- and site-matched control fibroblasts, could be reprogrammed to iPSCs. To establish this, we used Sendai virus to successfully reprogram six primary fibroblast cell lines derived from ulcerated skin of two DFU patients (DFU8, DFU25), nonulcerated foot skin from two diabetic patients (DFF24, DFF9), and healthy foot skin from two nondiabetic patients (NFF12, NFF14). We confirmed reprogramming to a pluripotent state through three independent criteria: immunofluorescent staining for SSEA-4 and TRA-1-81, formation of embryoid bodies with differentiation potential to all three embryonic germ layers in vitro, and formation of teratomas in vivo. All iPSC lines showed normal karyotypes and typical, nonmethylated CpG sites for OCT4 and NANOG. iPSCs derived from DFUs were similar to those derived from site-matched nonulcerated skin from both diabetic and nondiabetic patients. These results have established for the first time that multiple, DFU-derived fibroblast cell lines can be reprogrammed with efficiencies similar to control fibroblasts, thus demonstrating their utility for future regenerative therapy of DFUs.
  • 机译 芳烃受体内源活性的阻断改善了鸡原始生殖细胞的体外维持
    摘要:Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which, in recent years, encouraged a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining whether addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by the blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion, and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells.
  • 机译 玻璃化解冻的胚胎衍生的兔胚胎干细胞
    摘要:The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model.
  • 机译 决定猪体细胞核转移效率的因素:超过200,000个重建胚的数据分析
    摘要:Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion, this is the first report of large-scale analysis of porcine cell nuclear transfer that provides important data for potential industrialization of HMC technology.
  • 机译 p53和ASF1A在绵羊肾脏细胞重编程为多能细胞中的作用
    摘要:Since the first report of induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka, numerous attempts have been made to derive iPSCs from other species via the ectopic expression of defined factors. Sheep iPSCs (siPSCs) have significant potential for biotechnology and agriculture. Although several groups have described siPSCs, the reprogramming efficiency was extremely low. The exogenous transgenes could be not silenced in the iPSCs, which hampered their development and application. Here, we report that p53 knockdown and antisilencing function 1A (ASF1A) overexpression promoted iPSC generation from sheep kidney cells (SKCs). Compared with transduction with eight human defined transcription factors (Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, hTERT, and SV40LT), the additional introduction of p53 RNA interference (RNAi) and/or ASF1A in the presence of small-molecule compounds [vitamin C (Vc) and valproic acid (VPA)] greatly improved the efficiency of sheep iPSC generation. The siPSCs exhibited morphological features similar to mouse embryonic stem cells (ESCs) and were positive for alkaline phosphatase and, pluripotent marker genes (Oct4, Nanog, Sox2, Rex1, TRA-1-60, TRA-1-81, and E-cadherin). Furthermore, these cells exhibited a normal karyotype of 54 chromosomes and were able to differentiate into all three germ layers both in vitro and in vivo. Moreover, the exogenous genes were silenced in siPSCs when p53 small hairpin RNA (shRNA) and ASF1A were added. Our results may help to reveal the role of p53 and ASF1A in sheep somatic cell reprogramming and provide an efficient approach to reprogramming sheep somatic cells.

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