Male germ line stem cells: from cell biology to cell therapy

Research using stem cells has several applications in basic biology and clinical medicine. Recent advances in the establishment of male germ line stem cells provided researchers with the ability to identify, isolate, maintain, expand and differentiate the spermatogonia, the primitive male germ cells, as cell lines under in vitro conditions. The ability to culture and manipulate stem cell lines from male germ cells has gradually facilitated research into spermatogenesis and male infertility, to an extent beyond that facilitated by the use of somatic stem cells. After the introduction of exogenous genes, the spermatogonial cells can be transplanted into the seminiferous tubules of recipients, where the transplanted cells can contribute to the offspring. The present review concentrates on the origin, life cycle and establishment of stem cell lines from male germ cells, as well as the current status of transplantation techniques and the application of spermatogonial stem cell lines.     

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.     

胚胎干细胞及诱导多能干细胞在胚胎毒性研究中的应用

药物对于生殖细胞或者早期胚胎的影响将会引起不孕或者种植前胚胎的发育异常,进而引起胚胎毒性或者是后代的畸形,因此药物的临床应用需要有可靠的实验数据证明其对胚胎的影响,而胚胎干细胞(embryonic stem cell,ESC)由于其无限增殖及多向分化的潜能而作为研究药物胚胎毒性的细胞模型得到广泛应用,以ESC为基础的胚胎干细胞实验(embryonic stem cell test,EST)是获得国际认可的胚胎毒性评价的体外替代方法,但是该实验方法的快速性和准确性存在一定的局限性,目前该细胞模型的研究主要集中于快速性和准确性的优化。新兴的诱导多能干细胞(induced pluripotent stem cells,i PSC),由于具有与ESC相似的增殖和分化特性,目前也被逐步应用于药物胚胎毒性的研究。     

Vitamin A/Retinol and Maintenance of Pluripotency of Stem Cells

Retinol, the alcohol form of vitamin A is a key dietary component that plays a critical role in vertebrate development, cell differentiation, reproduction, vision and immune system. Natural and synthetic analogs of retinol, called retinoids, have generally been associated with the cell differentiation via retinoic acid which is the most potent metabolite of retinol. However, a direct function of retinol has not been fully investigated. New evidence has now emerged that retinol supports the self-renewal of stem cells including embryonic stem cells (ESCs), germ line stem cells (GSCs) and cancer stem cells (CSCs) by activating the endogenous machinery for self-renewal by a retinoic acid independent mechanism. The studies have also revealed that stem cells do not contain enzymes that are responsible for metabolizing retinol into retinoic acid. This new function of retinol may have important implications for stem cell biology which can be exploited for quantitative production of pure population of pluripotent stem cells for regenerative medicine as well as clinical applications for cancer therapeutics.     

Spermatogonia: origin, physiology and prospects for conservation and manipulation of the male germ line

In recent years, the scientific community has become increasingly interested in spermatogonia. Methodological breakthroughs, such as germ cell transplantation and spermatogonial culture combined with novel germ line transfection strategies, have provided interesting new opportunities for studying the physiology of spermatogonial stem cells and their interaction with the stem cell niche. Furthermore, intense research into pluripotent and adult stem cells has generated new insight into the differentiation pathway of germ line stem cells and has opened new perspectives for stem cell technologies. The present review briefly introduces the physiology of spermatogonial stem cells and discusses future directions of basic research and practical approaches applicable to livestock maintenance and animal reproduction.     

Comparison of neurosphere cells with cumulus cells after fusion with embryonic stem cells: reprogramming potential

Embryonic stem cells (ESCs) are the pluripotent cells that also have the capacity to induce the genomic reprogramming of differentiated somatic cells. The progressively restricted genomic potential of somatic cells observed during embryonic development can be reverted to a pluripotent state by the formation of cell hybrids with ESCs. To assess the reprogramming potential of ESCs, we investigated the reprogramming of one of two different somatic cell populations, neurosphere cells (NSCs) and cumulus cells (CCs), after fusion with ESCs. Specifically, hybrid cells were produced by cell fusion of E14 ESCs with either NSCs or CCs containing the neo/lacZ and Oct4-GFP transgenes. The first reprogramming event, observed by the presence of Oct4-GFP in the hybrid cells, could be identified on Day 2, at approximately 45 h after fusion in both ESC-NSC and ESC-CC hybrids. In addition, the two ESC-somatic cell hybrids exhibit a similar reprogramming rate and share characteristics with the E14 ESC line: (1) expression of pluripotent markers (Oct4, Rex-1 and nanog); (2) inactivation of differentiated tissue-specific gene expression; and (3) the capacity to differentiate into all three germ layers. Taken together, our results suggest that the ESC-somatic cell hybrids have fully acquired ESC characteristics and that somatic cells of different tissue origin have the same potential to be reprogrammed after fusion with ESCs.     

Male germ cell transplantation: promise and problems

Male germ cell transplantation is a novel technique in which donor male stem germ cells are surgically transferred to the seminiferous tubules of a recipient testis by direct injection or via the rete testis or efferent duct. All germ cells that are destined to become stem spermatogonia are defined as male stem germ cells, including primordial germ cells from the gonadal ridges, and gonocytes and stem spermatogonia from the testis, all of which are transplantable and capable of undergoing normal spermatogenesis. Xenotransplantation of male germ cells from one species into the testis of another species, including human testicular cells in the mouse, has so far proved to be unsuccessful. However, the immunodeficient mouse testis can support rat spermatogenesis and produce apparently normal rat spermatozoa. The underlying mechanisms remain elusive. The present mini-review will focus on the importance of stem spermatogonial transplantation for testicular stem cell biology and discuss the likelihood of immune rejection after transplantation, which may limit the success of all male germ cell transplantation.     

Male germ cell transplantation in livestock

Male germ cell transplantation is a powerful approach to study the control of spermatogenesis with the ultimate goal to enhance or suppress male fertility. In livestock animals, applications can be expanded to provide an alternative method of transgenesis and an alternative means of artificial insemination (AI). The transplantation technique uses testis stem cells, harvested from the donor animal. These donor stem cells are injected into seminiferous tubules, migrate from the lumen to relocate to the basement membrane and, amazingly, they can retain the capability to produce donor sperm in their new host. Adaptation of the mouse technique for livestock is progressing, with gradual gains in efficiency. Germ cell transfer in goats has produced offspring, but not yet in cattle and pigs. In goats and pigs, the applications of germ cell transplantation are mainly in facilitating transgenic animal production. In cattle, successful male germ cell transfer could create an alternative to AI in areas where it is impractical. Large-scale culture of testis stem cells would enhance the use of elite bulls by providing a renewable source of stem cells for transfer. Although still in a developmental state, germ cell transplantation is an emerging technology with the potential to create new opportunities in livestock production.     

Nontraditional sources of pluripotent stem cells: a new chapter in the debate about embryonic stem cell research

Recent efforts to resolve the political impasse over human embryonic stem cells (ESC) have generated proposals for obtaining ESC while avoiding the destruction of human embryos. This new chapter in the scientific and ethical debate provides an important opportunity to introduce additional ethical considerations to enhance public discourse.     

Differentiation of embryonic and adult stem cells into insulin producing cells

Replacement of insulin producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans is successful in experienced centers. The wider application of this therapy, however, is limited by the lack of donor organs. Insulin producing cells generated from stem cells represent an attractive alternative. Stem cells with the potential to differentiate into insulin producing cells include embryonic stem cells (ESC) as well as adult stem cells from various tissues including the pancreas, liver, bone marrow and adipose tissue. The use of human ESC is hampered by ethical concerns but research with human ESC may help us to decipher important steps in the differentiation process in vitro since almost all information available on pancreas development are based on animal studies. The present review summarizes the current knowledge on the development of insulin producing cells from embryonic and adult stem cells with special emphasis on pancreatic, hepatic and human mesenchymal stem cells.     

干细胞向生殖细胞分化的研究进展

胚胎干细胞（ESC）是一种多能干细胞,具有向生殖细胞分化的能力,由于具有致瘤性和伦理问题,限制了ESC向临床应用发展的前景。成体干细胞（ASC）也是一类多能干细胞,理论上也能分化为生殖细胞,但诱导分化过程往往受到阻滞。诱导性多能干细胞（iPS）是一种具有多能性的重编程体细胞,一定程度上可替代ESC用于基础和临床研究。目前,全球不孕不育的发生率呈逐年递增的趋势,充分认识干细胞向生殖细胞分化的机制是解决该难题的重要理论前提。就各种干细胞向生殖细胞分化的研究进展作一综述。     

Somatische Stammzellen Grundlagen, Konzepte und Herausforderungen

Adult stem cells are multipotent cells that have the ability to self-renew and to differentiate into highly specialized cells. Differentiation of specific cells is not limited to early embryonic development. It also takes place within the adult organism. Adult stem are found in tissues and organs showing a high turn-over rate such as blood, skin or intestine. Moreover, tissues like liver or skin are able to regenerate after injury, indicating the presence of stem cells within these organs. Whereas blood stem cells are already used in transplantation medicine, the biology and therapeutic potential of stem cells isolated from other tissues are still under investigation. Currently, research is focused on the identification and analysis of the factors regulating stem cell self-renewal and differentiation. A better understanding of stem cell biology certainly will lead to new therapeutic concepts, i.e. the use of stem cells and/or their derivatives as replacement cells to treat diseases including for example diabetes, Parkinson's disease, spinal cord injury, stroke, burns, rheumatoid arthritis.     

干细胞向雌性生殖细胞分化的研究进展

干细胞是一类具有自我更新和多向分化潜能的细胞。根据其来源不同,干细胞可分为胚胎干细胞、成体干细胞以及诱导性多能干细胞。生殖细胞系负责跨代传递遗传和表观遗传信息,以确保新的正常个体产生。人类辅助生殖技术(ART)虽可解决部分难治性不孕不育患者的生育问题,但尚不能解决由于卵巢早衰生殖细胞缺乏导致的不孕,如果在体外可以将干细胞定向诱导分化为生殖细胞,则可能通过ART帮助卵巢早衰患者获得健康后代。雌性配子发育经历了多个严格、复杂的过程,包括原始生殖细胞(PGC)特化、增殖、迁移到生殖嵴并最终分化为成熟的卵母细胞。然而具体过程尚不明确。近年来学者已建立了干细胞向雌性生殖细胞分化的体外模型,并取得了长足进步。     

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.     

Stem cell sources for cardiac regeneration

Cell-based cardiac repair has the ambitious aim to replace the malfunctioning cardiac muscle developed after myocardial infarction, with new contractile cardiomyocytes and vessels. Different stem cell populations have been intensively studied in the last decade as a potential source of new cardiomyocytes to ameliorate the injured myocardium, compensate for the loss of ventricular mass and contractility and eventually restore cardiac function. An array of cell types has been explored in this respect, including skeletal muscle, bone marrow derived stem cells, embryonic stem cells (ESC) and more recently cardiac progenitor cells. The best-studied cell types are mouse and human ESC cells, which have undisputedly been demonstrated to differentiate into cardiomyocyte and vascular lineages and have been of great help to understand the differentiation process of pluripotent cells. However, due to their immunogenicity, risk of tumor development and the ethical challenge arising from their embryonic origin, they do not provide a suitable cell source for a regenerative therapy approach. A better option, overcoming ethical and allogenicity problems, seems to be provided by bone marrow derived cells and by the recently identified cardiac precursors. This report will overview current knowledge on these different cell types and their application in cardiac regeneration and address issues like implementation of delivery methods, including tissue engineering approaches that need to be developed alongside.     

Mammalian pre-implantation chromosomal instability: species comparison,evolutionary considerations,and pathological correlations

Abstract

Pre-implantation embryo development in mammals begins at fertilization with the migration and fusion of the maternal and paternal pro-nuclei, followed by the degradation of inherited factors involved in germ cell specification and the activation of embryonic genes required for subsequent cell divisions, compaction, and blastulation. The majority of studies on early embryogenesis have been conducted in the mouse or non-mammalian species, often requiring extrapolation of the findings to human development. Given both conserved similarities and species-specific differences, however, even comparison between closely related mammalian species may be challenging as certain aspects, including susceptibility to chromosomal aberrations, varies considerably across mammals. Moreover, most human embryo studies are limited to patient samples obtained from in vitro fertilization (IVF) clinics and donated for research, which are generally of poorer quality and produced with germ cells that may be sub-optimal. Recent technical advances in genetic, epigenetic, chromosomal, and time-lapse imaging analyses of high quality whole human embryos have greatly improved our understanding of early human embryogenesis, particularly at the single embryo and cell level. This review summarizes the major characteristics of mammalian pre-implantation development from a chromosomal perspective, in addition to discussing the technological achievements that have recently been developed to obtain this data. We also discuss potential translation to clinical applications in reproductive medicine and conclude by examining the broader implications of these findings for the evolution of mammalian species and cancer pathology in somatic cells.     

人胚胎干细胞建系研究中的伦理管理

目的：建立国人胚胎干细胞系递交国际干细胞库,并在此基础上建立既符合中国国情又得到国际认可的相关伦理管理体系。方法：在比尔盖茨基金会的资助下,与北京大学生命科学院再生生物学实验室合作,募集胚胎建立人胚胎干细胞系,在此过程中探讨可行的符合国际伦理原则的相关伦理管理机制。结果：成功建立了国人胚胎干细胞系及相关伦理管理体系。结论：进行干细胞研究时应充分重视伦理问题,国际干细胞伦理管理与中国相关伦理原则是可以有机结合的。     

人胚胎干细胞建系研究中的伦理管理

目的：建立国人胚胎干细胞系递交国际干细胞库,并在此基础上建立既符合中国国情又得到国际认可的相关伦理管理体系。方法：在比尔盖茨基金会的资助下,与北京大学生命科学院再生生物学实验室合作,募集胚胎建立人胚胎干细胞系,在此过程中探讨可行的符合国际伦理原则的相关伦理管理机制。结果：成功建立了国人胚胎干细胞系及相关伦理管理体系。结论：进行干细胞研究时应充分重视伦理问题,国际干细胞伦理管理与中国相关伦理原则是可以有机结合的。