鸡Ⅹ期囊胚细胞研究进展及其应用

鸡Ⅹ期囊胚细胞(Blastodermal cells,BCs)作为潜在的胚胎干细胞,在胚胎发育动物模型、禽类转基因技术以及家禽种质资源冷冻保存等方面有广阔的应用前景。文章对鸡BCs的形态特征、体外培养、诱导分化、遗传修饰、冷冻保存、性别鉴定等体外操作方面的研究与应用进行了综述。     

鸡生殖干细胞研究现状与展望

鸡生殖干细胞是一类多能性干细胞,既可以在体外进行长期的自我更新,又能保持多向分化潜能,在禽类胚胎发育研究、转基因禽类生产及家禽育种等方面均有巨大应用价值.本文主要就鸡生殖干细胞的分离、培养与鉴定方法的研究进展及其应用前景进行简要综述,为进一步发展更高效的鸡生殖干细胞培养体系并应用于生产实践提供一定的借鉴.     

禽类胚胎干细胞的研究进展

胚胎干细胞(embryonic stem cells,ES细胞)是一种具有无限增殖能力和全向分化潜能的细胞系,在胚胎发育分化、组织工程、转基因研究等方面具有重要的应用价值,是目前生命科学研究的热点之一.禽类产品是人类重要的蛋白质食物来源,开展禽类的胚胎干细胞研究,并将其用于生产实践,具有极其重要的意义.禽类ES细胞研究起步较晚,但进展较为迅速,许多学者已投入到禽类ES细胞的研究工作中.本文就禽类胚胎干细胞的建系、特征与鉴定及转基因操作等作一综述.     

胚胎干细胞的生物学特性及其应用研究

胚胎干细胞 (ES细胞 )是一类具有多向分化潜能的 ,在适合的条件下能在体外抑制分化培养 ,并能复制增殖的细胞。综述了胚胎干细胞的主要生物学特性及其在基因诱捕、转基因动物、疾病治疗等研究中的新进展和应用前景     

胚胎干细胞及种系嵌合体的研究进展

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     

鱼类胚胎干细胞的研究进展

胚胎干细胞是存在于早期胚胎或原始生殖嵴,具有发育全能性和无限增殖能力的一类细胞。作为实验材料,该类细胞已广泛应用于基因的表达与调控、细胞分化的机制、动物克隆及转基因动物生产等研究领域。与其他哺乳动物胚胎干细胞的研究相比,虽然鱼类胚胎干细胞的研究起步较晚,但近年来该领域的研究进展迅速。本文综述了鱼类胚胎干细胞的分离培养、生物学特性、体外分化能力及嵌合体形成等方面的研究进展。     

Nanog基因的生物学功能研究进展

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。     

胚胎干细胞的生物学特性及其应用研究

胚胎干细胞（ES细胞）是一类具有多向分化潜能的，在适合的条件下能在体外抑制分化培养，并能复制增殖的细胞。综述了胚胎干细胞的主要生物学特性及其在基因诱捕、转基因动物、疾病治疗等研究中的新进展和应用前景。     

鱼类胚胎干细胞的研究进展

胚胎干细胞是存在于早期胚胎或原始生殖嵴,具有发育全能性和无限增殖能力的一类细胞.作为实验材料,该类细胞已广泛应用于基因的表达与调控、细胞分化的机制、动物克隆及转基因动物生产等研究领域.与其他哺乳动物胚胎干细胞的研究相比,虽然鱼类胚胎干细胞的研究起步较晚,但近年来该领域的研究进展迅速.本文综述了鱼类胚胎干细胞的分离培养、生物学特性、体外分化能力及嵌合体形成等方面的研究进展.     

小鼠雌性多能干细胞X染色体的表观遗传状态研究进展

雌性多能干细胞(iPS细胞)多能性状态的获得伴随着X染色体的表观修饰重编程。分化终末状态的雌性体细胞中有1条X染色体发生异染色质化而导致其失活。在体细胞诱导多能性干细胞的过程中,失活的X染色体重新活化。在重编程过程中,多能因子和X染色体失活中心的非编码基因的联系紧密。文章主要从分子水平上讨论多能干细胞X染色体表观修饰的相关研究进展。小鼠胚胎干细胞(ES细胞)是标准的多能性基态。X染色体上非编码RNA的表达可能是一个评价iP S表观遗传状态的标记,相关研究可以为细胞治疗的临床应用提供重要依据。     

猪多潜能干细胞系的研究进展

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。     

Perspectives of germ cell development in vitro in mammals

Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are able to differentiate into all cell lineages of the embryo proper, including germ cells. This pluripotent property has a huge impact on the fields of regenerative medicine, developmental biology and reproductive engineering. Establishing the germ cell lineage from ESCs/iPSCs is the key biological subject, since it would contribute not only to dissection of the biological processes of germ cell development but also to production of unlimited numbers of functional gametes in vitro. Toward this goal, we recently established a culture system that induces functional mouse primordial germ cells (PGCs), precursors of all germ cells, from mouse ESCs/iPSCs. The successful in vitro production of PGCs arose from the study of pluripotent cell state, the signals inducing PGCs and the technology of transplantation. However, there are many obstacles to be overcome for the robust generation of mature gametes or for application of the culture system to other species, including humans and livestock. In this review, we discuss the requirements for a culture system to generate the germ cell lineage from ESCs/iPSCs.     

胚胎干细胞分离和克隆影响因素的研究进展

胚胎干细胞ES是从早期胚胎或原始生殖细胞中分离出来的能够在体外进行传代培养,而不分化的全能性细胞,ES细胞最主要的生物学特征是具有发育的全能性或多能性,ES细胞能在体外培养、增殖、传代、冻存和诱导分化,是研究生物发育、行为和建立人体疾病模型的理想工具,本文重点论述了影响胚胎干细胞分离和克隆的因素。并展望了胚胎干细胞的应用前景。     

Pluripotent stem cells--model of embryonic development,tool for gene targeting,and basis of cell therapy

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.     

Of stem cells and germ cells

Stem cells have an intrinsic capacity to self-renew and can differentiate to at least one specialized cell type. Different types of stem cells exist that can be cultured in vitro. The identity of the stem cells is marked by their origin and differentiation potential. Germ cells have similarities with pluripotent stem cells but are of a special order: They do not self-renew and are already differentiated, but they have the capacity to form a complete new organism after fertilization. This review focuses on pluripotent stem cells and discusses possibilities of generating pluripotent stem cells from germ cell precursors and possibilities of generating germ cells from stem cells. As it accompanies a plenary lecture at the 15th annual ESDAR Conference 2011, the overview is focused on stem cells from farm animal species and on results from my own research group.     

Interspecies cell fusion between mouse embryonic stem cell and porcine pluripotent cell

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.     

鸡胚胎干细胞及其应用研究进展

鸡胚胎干细胞是一种多能性干细胞,从X期胚盘分离胚盘细胞或早期鸡胚的生殖嵴分离原始生殖细胞,经体外长期抑制分化培养可得到鸡胚胎干细胞。为维持细胞在培养过程中的未分化状态,需要采用饲养层细胞培养,同时设计合理的培养液配方并添加多种抑制分化或促进增殖的细胞因子。通过碱性磷酸酶活性检测、胚胎表面特异性抗原检测、分化试验及嵌合体试验等方法,可对鸡胚胎干细胞进行准确鉴定。文章主要就鸡胚胎干细胞的分离、培养与鉴定方法的研究进展及其应用前景进行简要综述,为进一步发展更高效的鸡胚胎干细胞培养体系并应用于生产实践提供一定的借鉴。     

山羊雄性胎儿生殖细胞建立干细胞系初探

体外培养山羊50~68日龄雄性胎儿生殖细胞,并检测它们的碱性磷酸酶(AP)活性和Oct-4蛋白,探讨性别分化后的生殖细胞用于建立干细胞系的可行性及检测指标。当山羊胎儿睾丸细胞体外培养时,生殖细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性,其中有部分细胞克隆表现为AP假阳性。山羊胎儿生殖细胞克隆呈隆突状生长,多为圆形,与周围细胞界限分明,但克隆内细胞间界限不清。细胞克隆至少可以培养3代以上。研究结果显示,山羊雄性胎儿生殖细胞可以用于建立生殖系来源的干细胞系;AP和Oct-4蛋白不适宜用来检测体外培养的山羊胎儿生殖细胞及其来源的细胞系。     

Isolation and culture of rabbit primordial germ cells

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.     

Production of somatic chimera chicks by injection of bone marrow cells into recipient blastoderms

Several types of cells, including blastoderm cells, primordial germ cells, and embryonic germ cells were injected into early-stage recipient embryos to produce chimera avians and to gain insights into cell development. However, a limited number of studies of avian adult stem cells have also been conducted. This study is, to the best of our knowledge, the first to evaluate chicken bone marrow cells' (chBMC) ability to differentiate into multiple cell lineages and capability to generate chimera chicks. We induced random differentiation of chBMCs in vitro and injected immunologically selected pluripotent cells in chBMCs into the blastoderms of recipient eggs. The multipotency of BMCs from the barred Plymouth rock (BPR) was confirmed via AP staining, RT-PCR, immunocytochemistry, and FACS using specific markers, such as Oct-4 and SSEA-1, 3 and 4. Isolated chBMCs were found to be able to induce in vitro differentiation to multiple cell lineages. Approximately 5,000 chBMCs were injected into the blastoderms of white leghorn (WL) recipients and proved able to contribute to the generation of somatic chimera chicks with a frequency of 2.7% (2 of 73). Confirmation of chimerism in hatched chicks was achieved via PCR analysis using D-loop-specific primers of BPR and WL. Our study demonstrated the successful production of chimera chicks using chBMC. Therefore, we propose that the use of adult chBMCs may constitute a new possible approach to the production of chimera poultry, and may provide helpful studies in avian developmental biology.