From teratocarcinomas to embryonic stem cells

The recent derivation of human embryonic stem (ES) cell lines, together with results suggesting an unexpected degree of plasticity in later, seemingly more restricted, stem cells (so-called adult stem cells), have combined to focus attention on new opportunities for regenerative medicine, as well as for understanding basic aspects of embryonic development and diseases such as cancer. Many of the ideas that are now discussed have a long history and much has been underpinned by the earlier studies of teratocarcinomas, and their embryonal carcinoma (EC) stem cells, which present a malignant surrogate for the normal stem cells of the early embryo. Nevertheless, although the potential of EC and ES cells to differentiate into a wide range of tissues is now well attested, little is understood of the key regulatory mechanisms that control their differentiation. Apart from the intrinsic biological interest in elucidating these mechanisms, a clear understanding of the molecular process involved will be essential if the clinical potential of these cells is to be realized. The recent observations of stem-cell plasticity suggest that perhaps our current concepts about the operation of cell regulatory pathways are inadequate, and that new approaches for analysing complex regulatory networks will be essential.     

Guiding embryonic stem cells towards differentiation: lessons from molecular embryology

Embryonic stem cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types, including germ cells. These qualities have made mouse embryonic stem cells a valuable resource for genetic manipulation of the mouse genome. In addition, they present a powerful system for the in vitro dissection of mammalian embryonic development. The recent isolation of human embryonic stem cells has raised a lot of interest for the potential of transposing our knowledge of lineage-specific differentiation of embryonic stem cells to cell-based therapy of human disease. Recent reports have provided insights into the specific differentiation of embryonic stem cells to different cell types of the embryo. However, progress in this direction seems to depend on the knowledge of the mechanisms controlling lineage decisions during embryogenesis.     

A glimpse into molecular mechanisms of embryonic stem cells pluripotency: Current status and future perspective

Embryonic stem cells have potential differentiation ability into a large variety of cell lineages and proved to be an effective therapeutic modality. However, prolonged in vitro and ex-vivo expansions impair embryonic stem cells multipotentiality, and thereby limit their clinical application. In the past few years, research collected attempts to explore new insights into the molecular mechanisms participate in the stemness capacity of embryonic stem cells. Along with these comments, modalities and strategies with the potential to maintain embryonic stem cells multipotentiality are of great interest. In this review, the authors attempted to discuss the pathways participating in the preservation of embryonic stem cells multipotentiality and emphasized the novel strategies that help to harness regenerative potential.     

Regulation of p53 in embryonic stem cells

Despite an increasing interest in the role of the p53 tumour suppressor protein in embryonic stem cells, not much is known about its regulation in this cell type.We show that the relatively high amount of p53 protein correlates with a higher amount of p53 RNA in ES cells compared to differentiated cells. Moreover, p53 RNA is more stable in embryonic stem cells and the p53 protein is more often transcribed. This is at least partly due to decreased expression of miRNA-125a and 125b in embryonic stem cells. Despite its cytoplasmic localisation, p53 is degraded in 26S proteasomes in embryonic stem cells. This process is controlled by Mdm2, the deubiquitinating enzyme Hausp and Ubc13. In contrast, the E3 ligase PirH2 appears to be less important for the control of p53 in embryonic stem cells. During differentiation, p53 protein and RNA levels are decreased which corresponds to increased expression of miRNA-125a and miRNA-125b.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

Shaping the eye from embryonic stem cells: Biological and medical implications

Organogenesis is regulated by a complex network of intrinsic cues, diffusible signals and cell/cell or cell/matrix interactions that drive the cells of a prospective organ to differentiate and collectively organize in three dimensions. Generating organs in vitro from embryonic stem (ES) cells may provide a simplified system to decipher how these processes are orchestrated in time and space within particular and between neighboring tissues. Recently, this field of stem cell research has also gained considerable interest for its potential applications in regenerative medicine. Among human pathologies for which stem cell-based therapy is foreseen as a promising therapeutic strategy are many retinal degenerative diseases, like retinitis pigmentosa and age-related macular degeneration. Over the last decade, progress has been made in producing ES-derived retinal cells in vitro, but engineering entire synthetic retinas was considered beyond reach. Recently however, major breakthroughs have been achieved with pioneer works describing the extraordinary self-organization of murine and human ES cells into a three dimensional structure highly resembling a retina. ES-derived retinal cells indeed assemble to form a cohesive neuroepithelial sheet that is endowed with the intrinsic capacity to recapitulate, outside an embryonic environment, the main steps of retinal morphogenesis as observed in vivo. This represents a tremendous advance that should help resolving fundamental questions related to retinogenesis. Here, we will discuss these studies, and the potential applications of such stem cell-based systems for regenerative medicine.     

Generation of insulin-expressing cells from mouse embryonic stem cells

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.     

Human embryonic stem cells: Problems and perspectives

Generation of human embryonic stem cell lines is one of the most important achievements in biological science in the 20th century. It has excited a wide scientific and social response, as embryonic stem cells (ESC) may, in the future, be regarded as an unlimited source of transplantation materials for replacement cell therapy. ESC lines are derived, cultured, inner cell mass from human blastocysts is used in the in vitro fertilization procedure. To date, human embryonic cell lines have been obtained in more than 20 countries. In our country, embryonic stem cell research is carried out in the Institute of Cytology, Russian Academy of Sciences and the Institute of Gene Biology, Russian Academy of Sciences. Studies with human ESC go in several directions. Much attention is paid to finding the most optimal conditions for ESC cultivation, mainly to the development of cultivation techniques excluding animal feeder cells and other components of animal origin. Another direction is a large-scale analysis of gene expression specific to the embryonic state of cells and the corresponding signaling pathways. Great efforts are being focused on the directed differentiation of ESC into various tissue-specific cells. It has been shown that in vitro ESC are able to differentiate into virtually any somatic cells. Works are in progress to develop methods for “therapeutic cloning,” i.e. the transfer of somatic nuclei into enucleated oocytes or embryonic stem cell cytoblasts and their reactivation. Of great importance is the standardization of the human ESC lines. However, standard requirements for cells utilized for research or therapeutic purposes may be different. It has been found that many permanent human ESC lines underwent genetic and epigenetic variations. Therefore, the cell line genetic stability should be periodically verified. The main purpose of the review is to provide a detailed consideration of research on the genetic stability of human and mouse ESC lines. Human ESC lines established both in our country and others could not thus far be used in clinical practice. It is highly probable that undifferentiated ESCs cannot be applied for therapeutic purposes, as there is a risk of their malignant transformation. Therefore, main efforts should be focused on the production ESC progenitor and highly differentiated cells suitable for transplantation.     

Anthropological and ethical reflections on the production and use of embryonic stem cells

Abstract.  Stem cells and their potential therapeutic application have generated tremendous public interest, great enthusiasm among researchers and intense commercial interest. There are diverse sources of stem cells. According to their origin and their biological characteristics, they are classified as embryonic stem cells, germline stem cells and tissue stem cells. Until now, the most concrete therapeutic results have come from some adult tissue stem cells, with promising prospects also being offered by umbilical cord stem cells. Regarding embryonic stem cells, there is concern that they would be difficult to control in vivo . Nonetheless, many researchers are still pursuing their potential uses, convinced that they will be useful not only for study, but also for therapy, especially as a result of their high capacity for self-renewal as well as their broad potential for differentiation. This discussion which is eminently scientific in nature, and not lacking in ethical and political repercussions, will not be entered into above all regarding the allocation of available intellectual and economic resources.     

The action behind the words: embryonic stem cell research marches on

Talk of policy has dominated talk of science for those interested in embryonic stem cell science. But research is continuing, and the advances are making clear why embryonic stem cells are such an important scientific and medical resource.     

Murine embryonic stem cells as a model for human embryonic stem-cell research

Over the last several decades, murine embryonic stem cells (mESCs) have been used as a model for human embryonic stem cell (hESC) research. The relevance of this approach has not yet been proven. There is a great deal of evidence that is indicative of substantial differences between these two cell types. An analysis of the literature shows that the differences concern ESC proliferation, self-renewal, and differentiation. Consequently, mESC may be considered as a model object for hESC studies only for some aspects of their biology. The alternative model objects, such as primate ESC, are also discussed briefly in this review.     

Potential of embryonic and adult stem cells in vitro

Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.     

人胚胎神经干细胞的基础及应用研究进展

神经干细胞是中枢神经系统中具有增殖、自我更新能力以及多种分化潜能的细胞,对它的研究已经成为神经生物学、发育生物学以及脑科学研究的一个热点。随着神经干细胞（特别是胚胎神经干细胞）的分离、培养成功,神经干细胞移植已被尝试用于神经系统损伤等疾病的治疗。但是,关于胚胎神经干细胞的研究尚处于初级阶段,特别是人胚胎神经干细胞的研究、报道还比较少。本文对国内、外近几年来关于人胚胎神经干细胞的基础及应用研究进展作了综述。     

Cancer genes hypermethylated in human embryonic stem cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.     

Derivation of human embryonic stem cell lines

Human embryonic stem cells (hESC) are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely, while retaining their capability to differentiate into specialized somatic cell types. Over the last decade there has been great interest in derivation and culture of these cells, as they can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation in humans, as well as for other medical uses such as drug discovery. The source of hESC lines is usually excess human embryos from in vitro fertilization treatments, although novel ways of producing hESCs have been suggested recently. The actual methods of hESC derivation have not changed greatly since the first report by Thomson et al. in 1998 . However, the main emphasis over the last several years has been in finding defined conditions for derivation and culture of hESCs, because to enable the clinical use of hESC for cell transplantation, the use of animal derived biological components is no longer acceptable. For basic research, the aim is to replace even human derived materials with completely defined systems. In this paper we describe methods utilized in our laboratory for hESC derivation and describe two studies conducted in an attempt to improve derivation efficiency and to enable research outcomes to be achieved using fewer embryos.     

The importance of valid disclosures in the human embryonic stem cell research debate

Abstract.  Misinformation erodes the legitimacy of any public debate. Since the start of human embryonic stem cell research deliberations in the USA, misinformation concerning the nature of human embryos, their availability for research, and the potential for using them to develop new medical therapies have been widespread and persistent. Basic facts, well understood by physicians and biologists, have been so misstated and misrepresented in the news media and political speeches that the general public has been put in a state of constant uncertainty. The solution to the present troubling condition is better education in the form of diligent, honest, and complete scientific disclosure by responsible scientists and physicians; and more care given to accurate reporting by news media. Several key aspects of newly emerging embryonic and non-embryonic stem cell technologies are defined and discussed as they relate to the debate over the use of human embryos for medical research. An important topic for consideration is how to disclose with clarity the scientific basis for human embryonic life. Thereafter, failings in proposed technologies for developing new therapies with human embryonic stem cells, that have been grossly under-reported, are examined. Finally, properties of adult stem cells are presented in contradistinction to embryonic stem cells, both in terms of adult stem cells as a scientifically better alternative to embryonic stem cells and in terms of the technological challenges that must be overcome to realize the potential of adult stem cells for new medical therapies.