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1.
胚胎干细胞及种系嵌合体的研究进展 总被引：1，自引：0，他引：1
胚胎干细胞是着床前的囊胚内细胞团或早期胎儿的原始生殖细胞经体外分化抑制培养建立的多能性细胞系 ,具有与胚胎细胞相似的形态特征和分化潜能 ,体外培养时保持未分化状态 ,可以传代增殖。改变维持胚胎干细胞不分化的培养条件 ,胚胎干细胞可自发分化成多细胞结构。在一定诱导下 ,胚胎干细胞可向多个方向分化 ,并生成多种功能细胞。胚胎干细胞注入到胚泡期胚胎或与桑椹期胚胎聚合 ,可以参与包括性腺在内的各种组织的嵌合体的形成。胚胎干细胞在细胞分化与调控 ,胚胎发育 ,遗传病 ,肿瘤 ,免疫和组织或器官移植等研究中显示着广泛的应用前景。而种系嵌合体的获得是实现 ES细胞途径的决定步骤 ,低的种系嵌合率则是制约 ES细胞应用的关键。提高供体 PGCs在受体生殖腺中的比例 ,缩短 ES细胞的体外培养时间 ,以及注入早期发育阶段的受体胚胎等都能提高种系嵌合率。文章从多个方面综述了胚胎干细胞的最新研究成果 ,并着重以禽类 ES细胞为例论述了种系嵌合体的检测方法 ,种系嵌合率的影响因素以及提高种系嵌合率的方法 相似文献
2.
Gerelchimeg Bou Jia Guo Yuan Fang Xuechun Li Renyue Wei Yan Li Zhonghua Liu 《Reproduction in domestic animals》2021,56(8):1095-1103
In the area of stem cell research, fusion of somatic cells into pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell on the pluripotent state of somatic nucleus. As many other groups, we previously established a porcine pluripotent cell line at a low potential. Therefore, here, we performed experiments to investigate if the fusion with mouse ES cell could improve the pluripotent state of porcine pluripotent cell. Our data showed that resultant mouse–porcine interspecies fused cells are AP positive, and could be passaged up to 20 passages. Different degrees of increases in expression of porcine pluripotent genes proved that pig-origin gene network can be programmed by mouse ES. Further differentiation study also confirmed these fused cells’ potential to form three germ layers. However, unexpectedly, we found that chromosome loss and aberrant (especially in porcine chromosomes) is severe after the cell fusion, implying that interspecies cell fusion may be not suitable to study porcine pluripotency without additional supportive conditions for genome stabilization. 相似文献
3.
Recent Progress in Embryonic Stem Cell Research and Its Application in Domestic Species 总被引：1，自引：0，他引：1
TAL Brevini S Antonini G Pennarossa F Gandolfi 《Reproduction in domestic animals》2008,43(S2):193-199
Many reports described cell lines derived in domestic species, which presented several important features typical of embryonic stem cells (ESCs). Such features unfortunately did not include the capacity to generate germ-line chimeras, therefore limiting the possibility to use these cells as tools for the genetic manipulation. However, farm animal ESCs may still be useful for the generation of transgenic animals as usually have a self-renewal capacity more prolonged than normal primary cultures thus increasing the possibility to transform and select cells to be used as nucleus donors in cloning procedures. Farm animal ESCs may also be an excellent experimental model in pre-clinical trials, assessing the feasibility of cell therapy because of the close morphological and physiological resemblance to humans of species like the pig. However, the persistent lack of standard methods for the derivation, maintenance and characterization of ESCs in domestic species stimulated the search for alternatives. Embryonic germ cells may represent such an alternative. Indeed, these cells showed a higher plasticity than ESCs as contributed to embryonic development forming chimeric newborns but, as for ESCs, standardization is still far away and efficiency is very low. Recent results indicated spermatogonial stem cells as possible tools for germ-line genetic modifications with some proof of principle results already achieved. But, a real break through could arrive from the multipotent germ-line stem cells, virtually equivalent to ESC, derived from newborn and adult mouse testis. 相似文献
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Pluripotent stem cells--model of embryonic development,tool for gene targeting,and basis of cell therapy 总被引：2，自引：0，他引：2
Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype. Murine ES cells are able to reintegrate fully into embryogenesis when returned into an early embryo, even after extensive genetic manipulation. In the resulting chimeric offspring produced by blastocyst injection or morula aggregation, ES cell descendants are represented among all cell types, including functional gametes. Therefore, mouse ES cells represent an important tool for genetic engineering, in particular via homologous recombination, to introduce gene knock-outs and other precise genomic modifications into the mouse germ line. Because of these properties ES cell technology is of high interest for other model organisms and for livestock species like cattle and pigs. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have yet been established for vertebrate species other than the mouse (Evans and Kaufman, 1981; Martin, 1981) and chicken (Pain et al., 1996). The in vitro differentiation capacity of ES cells provides unique opportunities for experimental analysis of gene regulation and function during cell commitment and differentiation in early embryogenesis. Recently, pluripotent stem cells were established from human embryos (Thomson et al., 1998) and early fetuses (Shamblott et al., 1998), opening new scenarios both for research in human developmental biology and for medical applications, i.e. cell replacement strategies. At about the same time, research activities focused on characteristics and differentiation potential of somatic stem cells, unravelling an unexpected plasticity of these cell types. Somatic stem cells are found in differentiated tissues and can renew themselves in addition to generating the specialized cell types of the tissue from which they originate. Additional to discoveries of somatic stem cells in tissues that were previously not thought to contain these kinds of cells, they also appear to be capable of developing into cell types of other tissues, but have a reduced differentiation potential as compared to embryo-derived stem cells. Therefore, somatic stem cells are referred to as multipotent rather than pluripotent. This review summarizes characteristics of pluripotent stem cells in the mouse and in selected livestock species, explains their use for genetic engineering and basic research on embryonic development, and evaluates their potential for cell therapy as compared to somatic stem cells. 相似文献
6.
Kanda A Sotomaru Y Shiozawa S Hiyama E 《The Journal of reproduction and development》2012,58(1):77-83
A number of mouse ES cells from inbred strains have been established to date, but efficiency varies across the different strains. The 129 strain mouse is efficient to establish, whereas C57BL/6 and BALB/c strains are not. It is possible that their genetic backgrounds account for the difference in their ability to establish ES cell lines. In this study, we attempted to establish C57BL/6J and BALB/c Cr ES cells by dual inhibition (2i) using two inhibitors (PD0325901 and CHIR99021) of extracellular signal regulated-kinase (ERK) and glycogen synthase kinase-3 (GSK-3), which promote ES cell differentiation. The results revealed that the establishment efficiencies of C57BL/6J and BALB/c Cr ES cells were remarkably increased by 2i. These ES cells stably expressed pluripotent markers and generated high-contribution chimeras with germline transmission. Furthermore, we generated germline chimeras from C57BL/6J ES cells through the method of gene modification. These findings indicate that 2i is a powerful tool for establishing C57BL/6J and BALB/c Cr ES cells with the ability to generate germline chimeras. 相似文献
7.
Isolation and culture of rabbit primordial germ cells 总被引：2，自引：0，他引：2
Kakegawa R Teramura T Takehara T Anzai M Mitani T Matsumoto K Saeki K Sagawa N Fukuda K Hosoi Y 《The Journal of reproduction and development》2008,54(5):352-357
Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells. 相似文献
8.
Minhua Hu Hengxi Wei Jingfeng Zhang Yinshan Bai Fenglei Gao Li Li Shouquan Zhang 《畜牧与生物技术杂志(英文版)》2013,4(1):12
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. 相似文献9.
猪多潜能干细胞是在体外建立起来的具有自我更新及三胚层分化潜能的一类干细胞,可应用于发育生物学研究、基因组编辑和疾病模型建立等各方面,在畜牧业生产及再生医学研究中具有重要的应用价值。培养体系对多潜能干细胞的成功构建具有重要意义,其包含多种成分,包括基础培养液、氨基酸等营养成分、小分子化合物(信号通路激活剂/抑制剂及细胞因子)共同维持细胞的多能性并抑制其分化。目前已有诸多关于猪多潜能干细胞的报道,但尚未获得高效的培养体系可以维持猪多潜能干细胞的长期传代并完成生殖嵌合。猪多潜能干细胞可分为原始态(Na6ve)、形成态(Formative)及始发态(Primed)3种不同多能性的状态特点,根据建系来源的不同分为猪扩展多潜能干细胞、猪胚胎干细胞、猪前原肠胚上胚层干细胞、猪诱导多能干细胞4种干细胞类型。作者综述了猪多潜能干细胞培养体系中常用的细胞因子和信号通路激活剂/抑制剂对猪多潜能干细胞的多能性和分化能力的影响和作用,为进一步建立具有真正生殖嵌合能力的Na6ve多能性的猪多潜能干细胞系提供研究思路。 相似文献
10.
囊胚注射转基因ES细胞制作嵌合体的研究 总被引：2，自引：0，他引：2
在小鼠胚胎成纤维细胞制作的饲养层上培养并成功的维持了携带LacZ基因的胚胎干细胞系（S8），在此基础上，以S8为供体细胞，以远交系昆明白小鼠3．5d胚胎为受体，通过显微注射法将供体细胞转移到受体的囊胚腔内，经过恢复培养，移植到代孕鼠昆明白雌鼠的子宫中；后代在嵌合体出生一周后进行判定。本试验用8～13代的S8细胞共注射胚胎597枚，经1～3h恢复培养，有585枚胚胎重新具有膨大的囊胚腔，细胞轮廓分明，滋养层细胞间连接也清晰可见，胚胎成活率为97％；胚胎移植后，代孕母鼠共移植胚胎228枚，经17～19d的妊娠期后，产仔37只（2只死胎），产仔率为16％；有35只仔鼠（雄鼠18只，雌鼠17只）存活到可以判断毛色，共获得8只S8细胞毛色嵌合体小鼠，嵌合体的产生率为21．6％。结果表明用S8细胞经囊胚注射后能够获得嵌合体，并且嵌合体明显发生了性偏离现象。本试验为国内利用囊胚注射携带LacZ基因的胚胎干细胞获得嵌合体小鼠的首例报道。 相似文献
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Jin-Gu NO Mi-Kyung CHOI Dae-Jin KWON Jae Gyu YOO Byoung-Chul YANG Jin-Ki PARK Dong-Hoon KIM 《The Journal of reproduction and development》2015,61(2):90-98
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 ChariotTM 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. 相似文献
13.
胚胎干细胞的研究与应用 总被引：9，自引：0，他引：9
胚胎干细胞（ES细胞）是由早期胚胎内细胞团或盈儿原始生殖细胞分离克隆出的具有发育全能性的细胞，是动物多种组织细胞的祖细胞。由于ES细胞与克隆动物、转基因动物、组织工程、临床克隆治疗和发育生物学、遗传学以及昨动物疾病模型等研究与应用的关系密切，引起广大学者的关注和兴趣。尤其是从1999年以来，人类ES细胞研究取得很大进展，人们渴望该技术尽快成熟，应用于临床医学克隆治疗，在世界范围内掀起了ES细胞的研究热潮。海峡两岸应组织多学科、多行业、多单位的科技工作者协同攻关，使该项研究尽快取得突破性进展。 相似文献
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Nanog基因的生物学功能研究进展 总被引：1，自引：0，他引：1
胚胎干细胞具有无限增殖能力和多向分化潜能决定了它在医学及生物学基础研究中具有巨大的应用潜力。探索维持胚胎干细胞特性的分子机制成为胚胎干细胞的生物学研究中的热点。研究发现与维持胚胎干细胞多能性相关的基因有Oct4、Nanog、Sox2等，其中Nanog是2003年5月末发现的一个基因，它对维持胚胎干细胞多能性起关键性作用，能够独立于L1F/Stats维持ICM和ES细胞的多能性。几年来，Nanog的生物学功能及其与Oct4、Sox2等多能性维持基因之间的相互作用关系已有较为深入的研究。作者在综述Nanog基因的表达特征和功能的基础上，重点探讨Nanog基因表达调控以及Oct4、Sox2等多能性维持基因之间的相互作用关系，并展望其应用前景。 相似文献
15.
Mouse spermatozoa can be freeze dried without losing genetic integrity and reproductive potential. However, it is not known if freeze-dried mouse cells similarly maintain their genetic integrity and developmental potential following nuclear transfer. Here, we investigated the developmental capacity and embryonic stem (ES) cell derivation of reconstructed oocytes by nuclear transfer using freeze-dried cumulus or ES cells. Cumulus and ES cells were lyophilized overnight and stored at 4 C for up to 1 week. After rehydration, all cells showed membrane damage and were unviable. However, following nuclear transfer, 1-4% of the reconstructed oocytes developed to the blastocyst stage. A total of five nuclear transfer ES (ntES) cell lines were generated from blastocysts and morulae. All ntES cell lines had normal karyotypes and were positive for the ES-cell-specific markers (alkaline phosphatase, Oct3/4 and Nanog). After aggregation of ntES cells with fertilized embryos, chimeric mice with a high level of coat color chimerism were generated. Our findings show that the genomic integrity of cells can be maintained after freeze-drying and that it is possible to produce offspring from the cells using nuclear transfer techniques. 相似文献
16.
Oliveira CS de Souza MM Saraiva NZ Tetzner TA Lima MR Lopes FL Garcia JM 《Reproduction in domestic animals》2012,47(3):428-435
Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels. 相似文献
17.
Casado JG Gomez-Mauricio G Alvarez V Mijares J Tarazona R Bernad A Sanchez-Margallo FM 《Veterinary immunology and immunopathology》2012,147(1-2):104-112
Mesenchymal stem cells have demonstrated their potentiality for therapeutic use in treating diseases or repairing damaged tissues. However, in some cases, the results of clinical trials have been disappointing or have not worked out as well as hoped. These disappointing results can be attributed to an inadequate or insufficient preclinical study. For medical and surgical purposes, the similarities between the anatomy of pig and human make this animal an attractive preclinical model. In this sense, for mesenchymal stem cell-based therapy, it is strongly necessary to have well characterized animal-derived mesenchymal stem cell lines to validate preclinical effectiveness of these cells. In this work, porcine mesenchymal stem cells (pMSCs) were isolated from bone marrow, adipose tissue and peripheral blood and compared in terms of differentiation potential, cell surface markers and gene expression. Our results demonstrated that the isolation and in vitro expansion protocols were feasible and effective. The data presented in this work are relevant because they provide an extensive phenotypic characterization; genetic study and differentiation behavior of the most commonly used stem cell lines for clinical practices. These pMSCs are widely available to scientists and could be a valuable tool to evaluate the safety and efficacy of adoptively transferred cells. 相似文献
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胚胎干细胞是未分化的具有增殖和自我更新能力的细胞,并且能分化成所有类型的体细胞以及生殖细胞。它们提供了早期胚胎分化的体外模型,也是基因操作的重要靶细胞。禽类多能性干细胞培养最重要的应用领域是以干细胞体外遗传修饰、鉴定为技术平台的家禽转基因技术。通过此技术对禽类基因进行遗传修饰与操作,在胚胎发育基础研究、转基因禽类生产及家禽育种等方面有巨大的应用前景。但是禽类多能性干细胞培养的许多基本问题仍亟待解决,如探索其建系的培养条件、揭示其维持多能性和增殖能力的分子机制等。文章综述了禽类多能性干细胞的分离方法、体外分化能力、嵌合体形成以及基因修饰方面的研究进展及目前的研究局限。 相似文献
20.
Wakayama T 《The Journal of reproduction and development》2007,53(1):13-26
Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications. 相似文献