Twenty years of embryonic stem cell research in farm animals

Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that an ESC can maintain an undifferentiated state indefinitely, self-renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the terminally differentiated cells of a conceptus. These attributes make the ESC an ideal source for genome editing for both agricultural and biomedical applications. Although, ESC lines have been successfully established from rodents and primates, authentic ungulate stem cell lines on the contrary are still not available. Outstanding issues including but not limited to differences in pluripotency characteristics among the existing ESC lines, pre-implantation embryo development, pluripotency pathways, and culture conditions plague our efforts to establish authentic ESC lines from farm animals. In this review, we highlight some of these issues and discuss how the recent derivation of induced pluripotent stem cells (iPSCs) might augur the establishment of robust authentic ESC lines from farm animals.     

Recent advances in stem and germ cell research: implications for the derivation of pig pluripotent cells

Pluripotent stem cells have the unique capacity to contribute to all the tissues of an adult animal after transfer into a host embryo. How pluripotency is acquired during early development and how it is maintained in stem cells have attracted the interest of many scientists for over three decades. Much progress in our understanding of how stem cells arise in culture and the signals required for homoeostasis has enabled the derivation of pluripotent cells in multiple species. Here, we discuss recent developments in stem cell biology that will impact the generation of pluripotent cells from different embryonic origins and will contribute to increase our capacity for generating transgenic animals.     

Recent Progress in Embryonic Stem Cell Research and Its Application in Domestic Species

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.     

Putative embryonic stem cell lines from pig embryos

Embryonic stem cells (ES cells) were first established in the mouse, and they represent a population of pluripotent, undifferentiated cells derived from early embryos that is capable of proliferating without any limitation in an undifferentiated state. These cells retain the ability to differentiate in vitro or in vivo into derivates of all three germ layers, and when injected into blastocysts, they can participate in the formation of all tissues, including gonads (germ-line chimeras). It is possible to transfect them with a gene of interest, and the resulting transgenic cell lines can also be used for production of chimeras. Unfortunately, mammalian germ-line chimeras that can carry an inserted gene into their progeny have only been produced in the mouse. Logically, before application of stem cell therapies into a human medicine, it is necessary to verify the efficiency and safety of these methods with an acceptable animal model. The pig is currently used as a very convenient animal for pre-clinical applications, and therefore establishment of porcine ES cell lines is highly needed; unfortunately, no convincing ES cell lines have been produced in this species (and other domestic animals) to date. In this article, we discuss the recent advances in this field, especially oriented on possible reasons and obstacles why derivation of porcine ES cell lines is still unsuccessful.     

Livestock induced pluripotent stem cells

Chimeric animals generated from livestock-induced pluripotent stem cells (iPSCs) have opened the door of opportunity to genetically manipulate species for the production of biomedical models, improving traits of agricultural importance and potentially providing a system to test novel iPSC therapies. The potential of pluripotent stem cells in livestock has long been recognized, with many attempts being chronicled to isolate, culture and characterize pluripotent cells from embryos. However, in most cases, livestock stem cells derived from embryonic sources have failed to reach a pluripotent state marked by the inability to form chimeric animals. The in-depth understanding of core pluripotency factors and the realization of how these factors can be harnessed to reprogram adult cells into an induced pluripotent state has changed the paradigm of livestock stem cells. In this review, we will examine the advancements in iPSC technology in mammalian and avian livestock species.     

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。     

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.     

Pestiviruses in wild animals

Pestiviruses are not strictly host-species specific and can infect not only domestic but also wild animals. The most important pestivirus, CSFV, infects domestic pigs and wild boars, which may cause a major problem for successful CSFV eradication programmes. Mainly BVDV specific antibodies have been reported in captive and free-living animals. Virus has been isolated from some of these animal species, but since BVDV can contaminate cell cultures and foetal calf serum, early reports of BVDV isolation have to be considered with caution. Genetic typing of early pestivirus isolates from wild species revealed that the majority were BVDV-1. Of the pestiviruses identified so far three species (CSFV, BVDV-1, giraffe pestivirus) and three genotypes (BDV-2, BDV-4, pronghorn) appear to circulate in wildlife animal populations. The potential for pestiviruses to spread between farm animals and free-living animals is discussed as are epidemiological and technical problems, and the future direction of research.     

Epigenetics and transgenerational inheritance in domesticated farm animals

Epigenetics provides a molecular mechanism of inheritance that is not solely dependent on DNA sequence and that can account for non-Mendelian inheritance patterns. Epigenetic changes underlie many normal developmental processes, and can lead to disease development as well. While epigenetic effects have been studied in well-characterized rodent models, less research has been done using agriculturally important domestic animal species. This review will present the results of current epigenetic research using farm animal models (cattle, pigs, sheep and chickens). Much of the work has focused on the epigenetic effects that environmental exposures to toxicants, nutrients and infectious agents has on either the exposed animals themselves or on their direct offspring. Only one porcine study examined epigenetic transgenerational effects; namely the effect diet micronutrients fed to male pigs has on liver DNA methylation and muscle mass in grand-offspring (F2 generation). Healthy viable offspring are very important in the farm and husbandry industry and epigenetic differences can be associated with production traits. Therefore further epigenetic research into domestic animal health and how exposure to toxicants or nutritional changes affects future generations is imperative.     

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.     

Male Germ Cell Transplantation

Transplantation of male germ line stem cells from a donor animal to the testes of an infertile recipient was first described in 1994. Donor germ cells colonize the recipient's testis and produce donor-derived sperm, such that the recipient male can distribute the genetic material of the germ cell donor. Germ cell transplantation represents a functional reconstitution assay for male germ line stem cells and as such has vastly increased our ability to study the biology of stem cells in the testis and define phenotypes of infertility. First developed in rodents, the technique has now been used in a number of animal species, including domestic mammals, chicken and fish. There are three major applications for this technology in animals: first, to study fundamental aspects of male germ line stem cell biology and male fertility; second, to preserve the reproductive potential of genetically valuable individuals by male germ cell transplantation within or between species; third, to produce transgenic sperm by genetic manipulation of isolated germ line stem cells and subsequent transplantation. Transgenesis through the male germ line has tremendous potential in species in which embryonic stem cells are not available and somatic cell nuclear transfer has limited success. Therefore, transplantation of male germ cells is a uniquely valuable approach for the study, preservation and manipulation of male fertility in animals.     

Development of New Stem Cell-based Technologies for Carnivore Reproduction Research

New reproductive technologies based on stem cells offer several potential benefits to carnivore species. For example, development of lines of embryonic stem cells in cats and dogs would allow for the generation of transgenic animal models, which could be used to advance both veterinary and human health. Techniques such as spermatogonial stem cell transplantation (SSCT) and testis xenografting offer new approaches to propagate genetically valuable individual males, even if they should die before producing sperm. These techniques might therefore have application to the conservation of endangered species of carnivores, as well as to biomedical research. Recently, our laboratory has successfully performed SSCT in the dog, with a recipient dog producing sperm of donor genetic origin. Testis xenografting has been used to produce sperm from pre-pubertal testis tissue from both cats and ferrets. These early steps reinforce the need not only for research on stem cell technologies, but also for additional research into complementary technologies of assisted reproduction in carnivores, so that the widest array of research and clinical benefits can be realized.     

精原干细胞移植在家畜生产中的应用

本文阐述了精原干细胞的分离纯化、冷冻保存、移植等一系列相关研究技术 ,同时围绕国内外最新研究进展展望了精原干细胞在家畜生产上的潜在应用     

Ethical stockmanship

The objective of this review is to consider the ethics of stockmanship, particularly from the perspective of the nature and extent of the duties of stockpeople to their farm animals. It will consider what science tells us about the impact of stockmanship on the animal, particularly the welfare of the farm animal. The effects of human-animal interactions on the stockperson will also be considered, since these interactions affect the work performance and job satisfaction of the stockperson and thus indirectly affect animal welfare. Animal ethics is broader than animal welfare and includes economic as well as philosophical, social, cultural and religious aspects. This paper is predicated on the view that farm animals can suffer, and that animal suffering is a key consideration in our moral obligations to animals. Housing and husbandry practices affect farm animal welfare and thus farmers and stockpeople have a responsibility to provide, at minimum, community-acceptable animal housing and husbandry standards for their animals. The farmer's or stockperson's attitudes and behaviour can directly affect the animal's welfare and thus they also have a responsibility to provide specific standards of stockmanship for these animals. However, research suggests that the behaviour of some stockpeople is not as correct as it might be. Such situations exemplify the inevitably unequal human - domestic animal relationship, and this inequality should be considered in analysing the boundary between right and wrong behaviour of humans. Thus ethical discussion, using science and other considerations and involving stockpeople, livestock industries, government and the general public, should be used to establish and assure acceptable stockperson competencies across the livestock industries. Training programs targeting the key attitudes and behaviour of stockpeople presently offer the livestock industries good opportunities to improve human-animal interactions.     

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     

Comparative Genomics Approach to Identify Candidate Genetic Loci for Male Fertility

The article presents multi‐species, genome‐wide, comparative approach to review male fertility‐associated loci to contribute to the development of new genetic markers that could be of interest for functional studies and have the potential to be implemented in farm animal breeding programmes. We reviewed 835 male fertility‐associated candidate loci from seven species and presented them as bovine orthologues where possible. The candidate loci were identified exploiting seven different research approaches: (i) data from animal models: mouse transgenics and knock‐outs (569 genes) and random chemical mutagenesis of mouse genome (31); (ii) animal QTL (69); (iii) genes differentially expressed between fertile and subfertile phenotype in humans and mouse (95); (iv) DNA sequence variations that show specific allele‐phenotype interactions (43 in human and 13 in farm animals); (v) germ line‐specific small non‐coding RNAs (47); (vi) testes expressed genes controlling complex differentiation process of mammalian spermatogenesis (6); and (vii) epigenetically regulated genes (4). According to the number of different research approaches reporting effects of individual genes, we selected 33 most promising candidate genes, which were further in silico analysed for expression levels in testes, genetic variability and top biological functions in functional networks. The aim of this study was to review systematically male fertility‐associated candidate loci using integrated information from different study approaches and species, which will further facilitate development of novel genetic markers for selection towards improved fertility in domestic animals.