The origin and identity of embryonic stem cells

Embryonic stem (ES) cells are used extensively in biomedical research and as a model with which to study early mammalian development, but their exact origin has been subject to much debate. They are routinely derived from pre-implantation embryos, but it has been suggested that the cells that give rise to ES cells might arise from epiblast cells that are already predisposed to a primordial germ cell (PGC) fate, which then progress to ES cell status via the PGC lineage. Based on recent findings, we propose here that ES cells can be derived directly from early epiblast cells and that ES cells might arise via two different routes that are dictated by their culture conditions.     

Challenges of primate embryonic stem cell research

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.     

Cardiac specification of embryonic stem cells

Over the past decade, cell transplantation has been recognized as a mean of repairing infarcted myocardium. Both adult stem cells and differentiated cells have yielded encouraging results with regard to engraftment into postinfarction scars. However, these cells now feature serious restrictions. Asan alternative, embryonic stem (ES) cells are particularly attractive, because of their plasticity and the subsequent possibility to drive them towards a cardiomyogenic phenotype after exposure to appropriate growth factors. An additional theoretical advantage of ES cells is their expected immune privilege. In this article, we summarize the findings obtained in cell therapy using ES cells and discuss the molecular mechanisms of cardiac specification of the cells.     

Isolation of epiblast stem cells from preimplantation mouse embryos

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.     

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.     

Derivation of keratinocyte progenitor cells and skin formation from embryonic stem cells

Despite numerous elegant transgenic mice experiments, the absence of an appropriate in vitro model system has hampered the study of the early events responsible for epidermal and dermal commitments. Embryonic stem (ES) cells are derived from the pluripotent cells of the early mouse embryo. They can be expanded infinitely in vitro while maintaining their potential to spontaneously differentiate into any cell type of the three germ layers, including epidermal cells. We recently reported that ES cells have the potential to recapitulate the reciprocal instructive ectodermal-mesodermal commitments, which are characteristic of embryonic skin formation. Derivation of epidermal cells from murine ES cells has been successfully established by exposing the cells to precisely controlled instructive influences normally found in the body, including extracellular matrix and the morphogen BMP-4. These differentiated ES cells are able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment similar to native skin. This bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling skin development and epidermal stem cell properties.     

DNA repair in murine embryonic stem cells and differentiated cells

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.     

Isolation and use of embryonic stem cells from livestock species

Abstract

Embryonic stem (ES) cells are pluripotent cells isolated from early embryos. They proliferate in culture and retain the capacity to differentiate both in vitro and In vivo, including contributing to chimeric tissues after injection into normal blastocysts. Over the past decade ES cells have been used extensively as a model for embryogenesis. More recently they have been shown to be capable of stable integration of exogenous DNA and used for numerous studies involving genomic manipulation. ES cells provide many opportunities for genetic engineering of domestic livestock species, but to date their isolation from embryos has been documented only for the mouse and perhaps the hamster. Efforts to isolate pluripotent ES cells from embryos of domestic livestock species are described, including some of the problems encountered.     

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.     

The liberation of embryonic stem cells

Mouse embryonic stem (ES) cells are defined by their capacity to self-renew and their ability to differentiate into all adult tissues including the germ line. Along with efficient clonal propagation, these properties have made them an unparalleled tool for manipulation of the mouse genome. Traditionally, mouse ES (mES) cells have been isolated and cultured in complex, poorly defined conditions that only permit efficient derivation from the 129 mouse strain; genuine ES cells have not been isolated from another species in these conditions. Recently, use of small molecule inhibitors of glycogen synthase kinase 3 (Gsk3) and the Fgf-MAPK signaling cascade has permitted efficient derivation of ES cells from all tested mouse strains. Subsequently, the first verified ES cells were established from a non-mouse species, Rattus norvegicus. Here, we summarize the advances in our understanding of the signaling pathways regulating mES cell self-renewal that led to the first derivation of rat ES cells and highlight the new opportunities presented for transgenic modeling on diverse genetic backgrounds. We also comment on the implications of this work for our understanding of pluripotent stem cells across mammalian species.     

Protecting genomic integrity in somatic cells and embryonic stem cells

Mutation frequencies at some loci in mammalian somatic cells in vivo approach 10(-4). The majority of these events occur as a consequence of loss of heterozygosity (LOH) due to mitotic recombination. Such high levels of DNA damage in somatic cells, which can accumulate with age, will cause injury and, after a latency period, may lead to somatic disease and ultimately death. This high level of DNA damage is untenable for germ cells, and by extrapolation for embryonic stem (ES) cells, that must recreate the organism. ES cells cannot tolerate such a high frequency of damage since mutations will immediately impact the altered cell, and subsequently the entire organism. Most importantly, the mutations may be passed on to future generations. ES cells, therefore, must have robust mechanisms to protect the integrity of their genomes. We have examined two such mechanisms. Firstly, we have shown that mutation frequencies and frequencies of mitotic recombination in ES cells are about 100-fold lower than in adult somatic cells or in isogenic mouse embryonic fibroblasts (MEFs). A second complementary protective mechanism eliminates those ES cells that have acquired a mutational burden, thereby maintaining a pristine population. Consistent with this hypothesis, ES cells lack a G1 checkpoint, and the two known signaling pathways that mediate the checkpoint are compromised. The checkpoint kinase, Chk2, which participates in both pathways is sequestered at centrosomes in ES cells and does not phosphorylate its substrates (i.e. p53 and Cdc25A) that must be modified to produce a G1 arrest. Ectopic expression of Chk2 does not rescue the p53-mediated pathway, but does restore the pathway mediated by Cdc25A. Wild type ES cells exposed to ionizing radiation do not accumulate in G1 but do so in S-phase and in G2. ES cells that ectopically express Chk2 undergo cell cycle arrest in G1 as well as G2, and appear to be protected from apoptosis.     

Using embryonic stem cells to introduce mutations into the mouse germ line

It is now possible, through the use of a number of experimental technologies, to transfer genetic information into mouse embryos to stably alter the genetic constitution of mice. This experimental approach, namely the generation of so-termed "transgenic" animals, is affording new insights into a wide variety of biological problems. This review focuses on one system for the generation of transgenic mice, which utilizes tissue culture cell lines of embryonic stem cells, termed ES cells. The remarkable property of ES cells is that they retain the potential to reform an embryo; when they are replaced inside a carrier embryo, they resume normal development and contribute to all the tissues of the live-born chimeric animal. Recent experiments, using a repertoire of gene transfer techniques, have shown that ES cells are amenable to a variety of experimental manipulations in tissue culture. Moreover, it has been demonstrated that these genetically altered cells can be transferred into the germ line of chimeric mice, thus allowing the production of unique strains of animals for study. The applications of the ES cell system are reviewed, with particular emphasis on their use for the generation of random insertional mutations using a retrovirally mediated mutagenesis approach. Finally, the use of ES cells in conjunction with the recently described technique of homologous recombination, or "gene targeting," is discussed. This technology allows the generation of animals carrying extremely precise genetic modifications of endogenous genes.     

Effect of genetic background on establishment of mouse embryonic stem cells.

We established 13 embryonic stem (ES) cell lines from 542 embryos crossed between various strains of mice: 10 lines from 129/Sv-ter embryos (10/48, 20.8%) and 3 lines out of other 9 combinations of intra- or inter-strain matings (1 from intracross of C57BL/6CrSlc, 1 from B6D2F1 x C57BL/6CrSlc, 1 from Yok:ddY x Slc:ICR). No ES cell line from 129/Sv-ter x Slc:ICR embryos suggests that ICR strain might have inhibitory genetic factor(s) for the ES cell formation. Some ES cell lines could be obtained from hybrids even if none or few lines from their parental strains, suggesting a heterosis effect can be expected for establishing ES cell lines in mice.     

Stability of genomic imprinting in embryonic stem cells: lessons from assisted reproductive technology

Imprinted genes are expressed predominantly or exclusively from one allele only. This mode of gene expression makes the regulation of imprinted genes susceptible to epigenetic insults, which may in turn lead to disease. There is compelling experimental evidence that certain aspects of assisted reproductive technology (ART) such as in vitro cell culture may have adverse effects on the regulation of epigenetic information in mammalian embryos, including the disruption of imprinted genes and epigenetic regulators. Moreover, in humans, disorders of genomic imprinting have been reported in children conceived by ART. The derivation and in vitro culture of embryonic stem (ES) cells are potential points of origin for epigenetic abnormalities. There is evidence that defects of genomic imprinting occur in mouse embryonic stem cells, with similar data now emerging in related studies in non-human primate and human ES cells. It is therefore pertinent to rigorously assess the epigenetic status of all stem cells and their derivatives prior to their therapeutic use in humans. Focusing on the stability of genomic imprinting, this review discusses the current evidence for epigenetic disruption in mammalian embryonic stem cells in light of the epigenetic disruption observed in ART-derived mammalian embryos.     

Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2–6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.     

鱼类的胚胎干细胞

胚胎干细胞(ES)是未分化的细胞培养物，来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下，ES细胞能分化成各种细胞类型，包括生殖细胞。这样，ES细胞就提供了一个有效的纽带，将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前，ES细胞技术仅见之老鼠，因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类，人们已做了大量的尝试。我们以青鳉(Oryzias latipes)作为建立鱼类ES细胞技术的模式，通过建立并应用无滋养层细胞的培养条件，获得了来自中期囊胚的ES细胞系。青鳉的ES细胞和老鼠的ES细胞有很多共同特征，如二倍体核型、分化潜力和形成嵌合体。因此，在鱼类建立和应用ES细胞技术是可能的。青鳉ES细胞的培养条件已成功地应用到其它鱼类如斑马鱼甚至海水鱼。本文旨在以青鳉为模式，综述获得和应用模式鱼和经济鱼ES细胞的主要进展和前景。