The Use of Transgenic Mouse Models in the Study of Male Infertility

Over the past few decades with the rapid advances in embryo and embryonic stem cell manipulation techniques, transgenic mouse models have emerged as a powerful tool for the study of gene function and complex diseases including male infertility. In this review we give a brief history of the development of tools for the production of transgenic mouse models. This spans the advances from early pronuclear injection to the use of targeted embryonic stem cells to produce gene targeted, conditional, and inducible knockout mouse models. Lastly we provide a few examples to illustrate the utility of mouse models in the study of male infertility.     

Genetic modification of somatic cells for producing animal models and for cellular transplantation

Great progress has been made in two technologies related to biomedical research: (1) manipulating the genomes of cells; and (2) inducing stem cells in culture to differentiate into potentially useful cell types. These technologies can be used to create animal models of human disease and to provide cells for transplantation to ameliorate human disease. Both embryonic stem cells and adult stem cells have been studied for these purposes. Genetically modified somatic cells provide another source of cells for creating animal models and for cellular transplantation.     

Circulating stem cells and tissue repair

Stem cells are defined as cells that have clonogenic, self-renewing capacities and the capability to differentiate into multiple cell lineages. Whereas embryonic stem cells are derived from mammalian embryos in the blastocyst stage and can generate terminally differentiated cells of all 3 embryonic germ layers, adult human stem cells are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue as a consequence of physiologic cell turnover or tissue injury. The traditional idea of organ-restricted stem-cell differentiation is now being challenged by the suggestion that adult stem cells retain developmental plasticity. Preclinical and clinical studies described in this review provide evidence that within the blood circulate not only progenitor cells that differentiate into hematopoietic cells, but also stem/progenitor cells which can participate in the homeostasis, repair and replacement of solid organ tissues. In addition to the occurrence of cell fusion, there are 4 suggested mechanisms of adult stem cell differentiation into solid organ cells. Preclinical data support these models particularly that of transdifferentiation as the most likely model, allowing stem/progenitor cells to differentiate across lineage, tissue, and germ layer boundaries. There is increasing evidence that we can manipulate in vivo circulating adult stem cells to repair or regenerate solid organ tissue, which offers potential clinical benefit in the treatment of many hereditary and acquired diseases.     

Perspectives on transgenic livestock in agriculture and biomedicine: an update

It has been 30 years since the first transgenic mouse was generated and 26 years since the first example of transferring the technology to livestock was published. While there was tremendous optimism in those initial years, with most convinced that genetically modified animals would play a significant role in agricultural production, that has not come to be. So at first sight one could conclude that this technology has, to a large extent, failed. On the contrary, it is believed that it has succeeded beyond our original expectations, and we are now at what is perhaps the most exciting time in the development and implementation of these technologies. The original goals, however, have drastically changed and it is now biomedical applications that are playing a central role in pushing both technical and scientific developments. The combination of advances in somatic cell nuclear transfer, the development of induced pluripotent stem cells and the completion of the sequencing of most livestock genomes ensures a bright and exciting future for this field, not only in livestock but also in companion animal species.     

Stammzellbasierte In-vitro-Modelle als Ersatz für Tiermodelle bei Toxizitäts- und Wirksamkeitsprüfungen

Regarding toxicity and efficacy tests of pharmacological and chemical substances (REACH legislation in Europe), there is a strong need to develop alternative methods for animal in vivo studies, in particular for human in vitro models. Here we present results from early phases of projects exploring the potential of embryonic stem cell models, with a special emphasis on embryo toxicity and neuronal stress.We have been able to demonstrate key functional read-outs of neural hESC models, in addition to representing mechanistic aspects which are characteristic for ischemia or excitotoxicity. There is agreement that these mechanisms underlie a variety of human neurodegenerative diseases. We discuss the possibilities to develop more precise endpoints on the molecular level and present an example of a protein biomarker signature emerging from a European FP6 project about embryo toxicity (www.reprotect.eu), employing murine and human embryonic stem cell models.     

Stem cell strategies for Alzheimer's disease therapy

We have found much evidence that the brain is capable of regenerating neurons after maturation. In our previous study, human neural stem cells (HNSCs) transplanted into aged rat brains differentiated into neural cells and significantly improved the cognitive functions of the animals, indicating that HNSCs may be a promising candidate for cell-replacement therapies for neurodegenerative diseases including Alzheimer's disease (AD). However, ethical and practical issues associated with HNSCs compel us to explore alternative strategies. Here, we report novel technologies to differentiate adult human mesenchymal stem cells, a subset of stromal cells in the bone marrow, into neural cells by modifying DNA methylation or over expression of nanog, a homeobox gene expressed in embryonic stem cells. We also report peripheral administrations of a pyrimidine derivative that increases endogenous stem cell proliferation improves cognitive function of the aged animal. Although these results may promise a bright future for clinical applications used towards stem cell strategies in AD therapy, we must acknowledge the complexity of AD. We found that glial differentiation takes place in stem cells transplanted into amyloid-( precursor protein (APP) transgenic mice. We also found that over expression of APP gene or recombinant APP treatment causes glial differentiation of stem cells. Although further detailed mechanistic studies may be required, RNA interference of APP or reduction of APP levels in the brain can significantly reduced glial differentiation of stem cells and may be useful in promoting neurogenesis after stem cell transplantation.     

Derivation characteristics and perspectives for mammalian pluripotential stem cells

Pluripotential stem cells have been derived in mice and primates from preimplantation embryos, postimplantation embryos and bone marrow stroma. Embryonic stem cells established from the inner cell mass of the mouse and human blastocyst can be maintained in an undifferentiated state for a long time by continuous passage on embryonic fibroblasts or in the presence of specific inhibitors of differentiation. Pluripotential stem cells can be induced to differentiate into all the tissues of the body and are able to colonise tissues of interest after transplantation. In mouse models of disease, there are numerous examples of improved tissue function and correction of pathological phenotype. Embryonic stem cells can be derived by nuclear transfer to establish genome-specific cell lines and, in mice, it has been shown that embryonic stem cells are more successfully reprogrammed for development by nuclear transfer than somatic cells. Pluripotential stem cells are a very valuable research resource for the analysis of differentiation pathways, functional genomics, tissue engineering and drug screening. Clinical applications may include both cell therapy and gene therapy for a wide range of tissue injury and degeneration. There is considerable interest in the development of pluripotential stem cell lines in many mammalian species for similar research interests and applications.     

Possibilities of investigation and clinical application of adult human stem cells

Multipotent adult tissue stem cells have high plasticity and transdifferentiation ability. The stem cell therapy can be the solution of curing many severe diseases such as osteogenesis imperfecta, hepatic failure, heart muscle damage after myocardial infarction, I-type diabetes, variety of central nervous system disorders such as brain injury, stroke, Parkinson's disease and other neurodegenerative disorders. Isolation of certain types of stem cells is solved nowadays, but low frequency of these cells and lack of special identification markers make their isolation and search more difficult. The more and more developed in vitro cell culture technologies, the widespread macromolecule amplification and examination techniques of molecular biology and the gene technology tools for genetic modification of stem (and other) cells contribute to research and therapeutic applications of stem cells. Transfer of new genetic material to stem cells and expression of the gene product in daughter cells--because missing or damaged genes can be replaced--is an exiting approach of the treatment of congenital (enzyme deficiencies) and acquired human diseases (cancers).     

Neurogenesis and neurodegenerative diseases in human

Acute and chronic neurodegenerative diseases are the most common neurological disorders in human and affect millions of individuals worldwide. While the specific clinical presentation varies among such diseases, their common feature is neural cell death. Yet, despite major advances in the understanding of neural cell death, effective treatment for these diseases remains one of the foremost challenges for medicine today. The transplantation of embryonic cells into the diseased brain in human has emerged out a mere theory and is possible as a practical application. This advancement, however, has raised important ethical, technical and immunological concerns. Studies have documented that neurogenesis occurs in the adult brain and that endogenous neural stem/progenitor cells (NSCs) respond to neurodegenerative diseases, suggesting that it might be possible for dead or injured neural cells to be replaced by endogenous NSCs. In this regard it is especially interesting to know the biological behaviors of endogenous NSCs in response to neurodegenerative diseases. Understanding the mechanisms underlying these changes could lead to the development of new strategies for treating neurological diseases using endogenous NSC pool.     

Germ cell transplantation for the propagation of companion animals, non-domestic and endangered species

The transplantation of spermatogonial stem cells between males results in a recipient animal producing spermatozoa carrying a donor's haplotype. First pioneered in rodents, this technique has now been used in several animal species. Importantly, germ cell transplantation was successful between unrelated, immuno-competent large animals, whereas efficient donor-derived spermatogenesis in rodents requires syngeneic or immuno-compromised recipients. Transplantation requires four steps: recipient preparation, donor cell isolation, transplantation and identifying donor-derived spermatozoa. There are two main applications for this technology. First, genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in production of transgenic spermatozoa. Transgenesis through the male germ line has tremendous potential in species in which embryonic stem cells are not available and somatic cell nuclear transfer and reprogramming pose several problems. Second, spermatogonial stem cell transplantation within or between species offers a means of preserving the reproductive potential of genetically valuable individuals. This might have significance in the captive propagation of non-domestic animals of high conservation value. Transplantation of germ cells is a uniquely valuable approach for the study, preservation and manipulation of male fertility in mammalian species.     

National risk signatures and human embryonic stem cell research in mainland China

The international development of human embryonic stem cell research has become closely tied to global bioethics, which places moral responsibility on stem cell researchers. This article argues that the development of bioethical regulation of human embryonic stem cell research is better understood by approaching the institutionalisation of bioethics in terms of risk perceptions of stem cell scientists. Eschewing approaches that understand bioethical risk as a mere matter of morality or as a social construct, this article emphasises the materiality and strategic reasoning of bioethical views on risks associated with human embryonic stem cell research. Such an approach allows the identification of forms of risk rooted in the everyday practice of Chinese human embryonic stem cell research, including moral risk (as a violation of cultural values), material risk (in relation to the distribution of material resources and wealth), political risk (in terms of the political economy of bioethics and public debate) and reputational risk (in terms of personal and national honour). Although this analysis builds on Tom Horlick-Jones's concept of risk signatures of new technologies, which emphasise the capacities of different technologies to engender and delimit the particular social and cultural interpretations of the risks they generate, the article reveals the existence of a certain global awareness among stem cell scientists of risk signatures. They display a creative and strategic awareness regarding the possible opportunities and constraints the risk signature of human embryonic stem cell research affords in their particular institutional context compared to those of others abroad and at home in different environments. The existence of this form of reflexivity requires recognition and methodological accommodation.     

胚胎干细胞向肝细胞的诱导分化研究

胚胎干细胞在体外一定条件下能够分化为与原代培养肝细胞表型相似,并表达部分成熟肝细胞功能的类肝细胞.尽管目前在诱导条件的优化、分化过程的调控及临床应用的安全性等方面仍面临一系列问题,但研究胚胎干细胞向肝系的诱导分化及纯化,为终末期肝病的细胞移植治疗、生物人工肝及药物代谢和毒理研究提供了丰富的细胞来源.     

People, other animals and health knowledges: towards a research agenda

By serving as experimental models for human disease, animals have been instrumental to constructing biomedical knowledge. On the other hand, animals themselves increasingly benefit from biomedical expertise and technologies, as patients in their own right. Healthy companion animals have recently come to be viewed explicitly as potential sources of human health, which contrasts with the potential for animals to injure people or transmit infectious disease. In studies of biomedical and other health knowledges, nevertheless, only the animal model role has been explored in any depth. In this review article, we sketch and discuss three research concerns that currently inform studies of biomedical knowledge: medicalization and biomedicalization; constructing biomedical knowledge; and a concern with heterogeneity. We conclude that a more comprehensive and nuanced account of contemporary societies will result from further consideration of the importance of animals for how people understand health.     

人类植入前胚胎发育研究进展

随着近年无创成像技术、分子及基因组技术的发展,人类植入前胚胎发育的研究取得了相应进展。非侵入性实时成像法观察人类胚胎发育过程中最初3次有丝分裂可以预测囊胚发育的成败。目前世界各地多家体外受精（IVF）临床中心开始使用胚胎镜观察胚胎的卵裂时间和胚胎发育的空间模式。人类植入前胚胎发育的特征是重编程,包括精卵原核融合、表观遗传重编程和修饰、母源转录的全面退化和早期人类胚胎基因组激活,普遍认为在人胚胎发育的第3天即4-至8-细胞期胚胎基因组激活达高峰。谱系分化研究人类胚胎发育早期在8-细胞期每个细胞是独立的,但没有证据表明这一期不同细胞已经发生定向分化为滋养外胚层（TE）或内细胞团（ICM）。关于人单个卵母细胞的高精度全基因组测序、人类卵母细胞以及早期胚胎不同发育阶段基因转录组的动态变化已有报道。     

Experimental and clinical applications of molecular cell biology in nutrition and metabolism.

Rapid advances in molecular biology have yielded important new techniques for understanding the cellular mechanisms of normal homeostasis and disease. In particular, molecular laboratory methodologies have become important investigative tools for nutritional studies. Detection techniques for specific DNAs, RNAs, and proteins allow direct examination of cellular regulation of protein expression in health and illness. Construction of transgeneic models by recent techniques of inserting foreign genes into experimental animals has provided novel models for studies of cellular metabolism. In addition, molecular biology has had impact on clinical nutrition and therapy. Molecular techniques not only allow for early diagnosis of many inborn genetic errors of metabolism, recombinant technology has also provided for large-scale production of proteins and hormones of potential therapeutic value. The possibility for direct gene therapies is also nearing reality. Hence, understanding the language of molecular biology and the recent developments in this field is not only of research interest, but is also of clinical relevance.     

Embryonic stem cell therapy in experimental stroke: host-dependent malignant transformation

INTRODUCTION: Therapeutic application of embryonic stem cells in neurodegenerative disorders like stroke is widely investigated in preclinical animal models. AIM: The authors studied the therapeutic potential of murine embryonic stem cells in two rodent models of stroke. METHODS: Undifferentiated and predifferentiated stem cells were implanted into the non-ischemic hemisphere of mice and rats following focal brain ischemia. The brains were analysed by immunohistochemistry and histology. The in vitro differentiation of the cells was checked by immunocytochemistry and Western-blot. RESULTS: After xenotransplantation in rats undifferentiated cells migrated along the corpus callosum towards the ischemic injury. Later stem cells differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. These experiments demonstrated a hitherto unrecognized adverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells. CONCLUSION: This observation raises serious concerns about safety provisions when the therapeutic potential of human embryonic stem cells is tested in preclinical animal models. The clinical trials are based on the positive outcome of the xenologous experiments.     

Banking on it: public policy and the ethics of stem cell research and development

If the therapeutic potential of stem cell-based therapies is ever realized, demand for stem cells and derivative tissues will be tremendous and will create new challenges for health care systems, especially publicly funded health care systems. We propose a framework for the ethical analysis of stem cell research and development that considers the welfare of communities, tissue recipients, and cell sources in relation to a range of stem cell production and distribution options. Ethical desiderata include: equitable access, maximized potential therapeutic benefit across demographic and disease groups, and reasonable cost. Other ethical priorities include the minimization of stem cell line and tissue wastage, risk of immune rejection, risk of transmitting diseases, the use of human embryos, and risk to those contributing source cells. We array plausible sources of stem cells and distribution strategies to characterize 12 potential models for producing and distributing cells and tissues in the future. We describe "personalized", "matched", and "universalized" models, and compare the ethical acceptability of these models. Popular and scientific discourses about stem cells typically emphasize personalized or matched stem cell distribution models. We show that universalized models may ultimately best serve the interest of taxpayers, communities and patients who hold high stakes in the therapeutic success of stem cell science. They are therefore highly worthy of scientific pursuit. This conclusion is provisional and the framework must be reapplied as scientific knowledge, technological capacity and ethical mores evolve.