Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.     

Efficient production of chimeric mice from embryonic stem cells injected into 4- to 8-cell and blastocyst embryos

Background

Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem (ES) cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES cells into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator (PMM), and trace the fate of the injected ES cells.

Results

We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly.

Conclusions

These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.     

Efficient production of chimeric mice from embryonic stem cells injected into 4- to 8-cell and blastocyst embryos

Background： Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem （ES） cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES ceils into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator （PMM）, and trace the fate of the injected ES cells. Results： We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly. Conclusions： These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.     

猪胚胎干细胞的分离克隆

本文从分化抑制物、培养基的选择内细胞团的分离,胚胎干细胞的分离及鉴定方法等方面系统地综述了猪胚胎干细胞分离克隆的研究进展;并对其未来的应用前景进行展望。     

ES细胞克隆动物技术研究进展

ＥＳ细胞克隆动物在提高良种动物繁殖率和保护动物遗传资源领域具有重要的作用,ＥＳ细胞核移植是生产克隆动物的重要方法,ＥＳ细胞克隆率低和克隆动物生活力低是其存在的主要问题。     

影响胚胎干细胞分离克隆的因素分析

本文就胚胎质量和胚胎类型、分化抑制物、培养基、传代时间和消化液等方面对胚胎干细胞分离克隆的影响进行了讨论。     

干细胞的研究概况及应用前景

介绍了干细胞的概念、分类。综述了干细胞研究概况及应用前景。     

哺乳动物体细胞重编程机制的研究进展

体细胞重编程面临的进程缓慢和效率低下等问题,限制了其向临床应用的转化,该过程涉及细胞内基因表达的变化,认识和调控DNA的表观修饰是成功重编程的关键。本文对体细胞重编程过程中基因表达的表观影响以及去分化和转分化的表观控制进行了阐述,并综述了通过表观遗传修饰或信号转导途径增强重编程效率或替代特殊重编程转录因子的物质,不仅有利于阐明体细胞重编程的机制,也为仅采用化学物质即可诱导出多能干细胞的研究提供参考。     

Recent progress in bovine somatic cell nuclear transfer

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full‐term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT.     

利用动物胚胎干细胞生产转基因动物研究进展

介绍了利用动物胚胎干细胞生产转基因动物的程序和方法,并阐述了利用胚胎干细胞生产转基因动物的意义。     

哺乳动物体细胞核移植胚胎发育中表观重编程的研究进展

体细胞核移植（somatic cell nuclear transfer,SCNT）是一种能将已分化的体细胞重编程为全能胚胎的繁殖生物技术,在良种扩繁、濒危物种保护和治疗性克隆等方面有着广泛的应用前景,但极低的克隆效率、克隆动物胎盘异常、出生后胎儿畸形等严重限制了该技术的实际应用。造成克隆效率低和胚胎发育异常的主要原因是供体核表观遗传重编程错误或不完全。1958年,将非洲爪蟾（Xenopus laevis）幼体肠细胞核移入去核卵母细胞,获得了第1例SCNT动物个体;1986年,通过电融合1个卵裂球与去核卵母细胞成功获得了3只存活的羔羊;1997年,将成年母羊的乳腺上皮细胞与去核卵细胞电融合,获得首个SCNT哺乳动物"多利",开启了克隆时代,目前牛、小鼠、山羊、猪、欧洲盘羊、家兔、家猫、马、大鼠、骡子、狗、雪貂、狼、水牛、红鹿、单峰骆驼、食蟹猴等相继成功克隆,其中最引人瞩目的是2018年食蟹猴的成功克隆。作者通过将SCNT胚胎与受精胚胎的发育进行对比,阐述了SCNT过程中DNA甲基化、组蛋白修饰、基因组印迹、染色体状态等的重编程过程和缺陷,并从表观修饰剂、组蛋白去甲基化酶、抑制Xist表达、补充鱼精蛋白和精子RNA方面探讨单独或联合消除表观遗传重编程障碍对克隆效率的影响。随着低样本量测序技术的发展和完善,人们能够在SCNT胚胎中检测到更详细的全基因组表观遗传修饰图谱,进一步揭示SCNT胚胎表观遗传重编程中的缺陷,为提高克隆效率提供了线索。通过上述内容的阐述,希望为后续开发联合消除多种表观遗传障碍而提高克隆效率的策略和思路。     

小鼠胚胎干细胞培养、克隆、分离、传代研究

对影响小鼠胚胎干细胞（Embryonic stem cells,ES细胞）培养、克隆、分离、传代效果的因素进行了探索研究。应用223枚昆明白小鼠胚胎和20枚129品系小鼠胚胎的研究结果表明，129品系小鼠胚胎比昆明白小鼠胚胎更适合作为ES细胞建系的材料，两者FS出现率差异显著（P＜0．05）；以DMEM＋10％NBS＋10％FCS为基础培养液，分别加入LIF、胰岛素、LIF＋SCF，极显著提高昆明白小鼠胚胎贴壁率，ICM生长率及F1、F2出现率（P＜0．01），而在DMEM＋10％NBS＋10％FCS＋LIF＋SCF为培养液，得到昆明白小鼠胚胎最高贴壁率、ICM生长率及传代率；4dpc胚胎传代情况显著好于3．5dpc胚胎（P＜0．05）。     

Molecular signature and colony morphology affect in vitro pluripotency of porcine induced pluripotent stem cells

Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.     

Comparative genomic analysis of eight Leptospira strains from Japan and the Philippines revealing the existence of four putative novel genomic islands/islets in L. interrogans serovar Lai strain 56601

Leptospirosis is one of the most widespread zoonotic diseases worldwide and can be considered an emerging health problem to both human and animal. Despite the importance of the disease, complete genome sequences are currently available for only three Leptospira interrogans strains: 56601, Fiocruz L1-130, and IPAV. Therefore, intra- and inter-species comparative genomic analyses of Leptospira are limited. Here, to advance current knowledge of the genomic differences within Leptospira species, next-generation sequencing technology was used to examine the genomes of eight L. interrogans strains belonging to six different serogroups isolated from humans and dogs in Japan and the Philippines. The genomic sequences were mapped to that of the reference strain, L. interrogans serovar Lai strain 56601. The results revealed the presence of four novel genomic islands/islets (GIs) in strain 56601. This study provides a deeper insight into the molecular basis and evolutionary perspective of the virulence of leptospires.     

Two genomic variations in the E1 region of canine adenovirus type 2 strains

We amplified the E1 region of canine adenovirus type 2 genomes by the polymerase chain reaction (PCR) and analyzed the PCR products by using eight restriction endonucleases. Restriction patterns of the E1 region cleaved with HaeIII and RsaI revealed two genomic variations among the canine adenovirus type 2 strains. Although the clinical significance of two distinct genotypes among the canine adenovirus type 2 strains is currently unknown, these genomic variations are well conserved among different strains in each genotype and suggest that the Japanese field strains, with reference to the E1 region, are different from the non-Japanese strains examined.     

Epiblast Cell Number and Primary Embryonic Stem Cell Colony Generation Are Increased by Culture of Cleavage Stage Embryos in Insulin

Human embryos for hESC derivation are often donated at the cleavage stage and of reduced quality. Poor quality embryos have lower efficiency for hESC derivation. However, cleavage stage mouse embryos develop into higher quality expanded blastocysts if they are cultured with insulin, suggesting that this approach could be used to improve hESC derivation from poor quality cleavage stage embryos. The present study used a mouse model to examine this approach. In particular we examined the effect of insulin on the number of epiblast cells in blastocysts on days 4, 5 and 6 using Oct4 and Nanog co-expression. Second we examined the effect of insulin on the frequency with which outgrowths can be derived from these. Finally, we tested whether prior culture in the presence of insulin results in blastocysts with increased capacity to generate ESC colonies. Culture of cleavage stage embryos with insulin increased the number of Oct4 and Nanog positive cells in blastocysts at all time points examined. Prior culture with insulin had no effect on outgrowths generated from blastocysts plated on days 4 or 5. However, insulin treatment of blastocysts plated on day 6 resulted in increased numbers of outgrowths with larger epiblasts compared with controls. 13% of insulin treated day 6 blastocysts produced primary ESC colonies compared with 6% of controls. In conclusion, treatment with insulin can improve epiblast cell number in mice leading to an increase with which primary ESC colonies can be generated and may improve hESC isolation from reduced quality embryos donated at the cleavage stage.     

哺乳动物体细胞克隆技术的应用前景

体细胞克隆的成功为农业、医学以及生物学基础研究提供了新的工具。体细胞核移植技术和转基因、胚胎干细胞等现代生物技术的相互渗透更是活跃。当前,尽管体细胞核移植效率仍然不甚理想,而且克隆后代成活率低、畸形率高,但人们还是对该技术的发展充满了憧憬,尤其希望借助这一手段在攻克一些长期困扰人类的疑难疾病上有所突破。作者则总结了体细胞核移植在上述各方面的应用价值,供同行参考。     

小鼠孤雌胚的体内体外发育研究

小鼠孤雌激活卵登攀上培养于ＣＺＢ溶液中可发育至孵出囊胚阶段,而且孤雌胚移植后可在受体子宫内附植发育。单倍体、二倍体孤雌胚发育至桑椹胚的能力相似,但发育于囊胚的能力,后得高于前者。孤雌囊胚体外发育至孵出的比率仅为２０％,但在卵巢切除的受体子宫内期发育至孵出囊胚的比率提高到７３．３％。与受精扩张囊胚相比,孤雌扩张囊肿成纤维细胞饲养层上很难贴壁生长,但延迟孤雌囊胚的内细胞团（ＩＣＭ）呆在细胞饲养层上增殖