A simple and efficient cryopreservation method for primate embryonic stem cells

Human embryonic stem (ES) cells have the potential to differentiate into all cell types. As these cells may be able to provide an unlimited cell source for transplantation therapies, it is necessary to establish reliable methods for their handling and manipulation, including human ES cell cryopreservation. Here, we report the development of a simple and efficient cryopreservation method for primate ES cell lines using vitrification in conventional cryovials. Using standard slow-rate cooling methods, the cryopreservation efficiency for cynomolgus monkey ES cell lines was approximately 0.4%, while that for a human ES cell line was virtually 0%. Primate ES cell lines, however, were successfully cryopreserved by the present vitrification method using conventional cryovials yielding a survival rate of about 6.5% for monkey ES cells and 12.2% for human ES cells. Vitrified ES cells quickly recovered after thawing and exhibited a morphology indistinguishable from non-vitrified cells. In addition, they retained a normal karyotype and continued to express ES cell markers after thawing. Thus, our vitrification ES cell cryopreservation method expands the utility of primate ES cells for various research and clinical purposes.     

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.     

小鼠孤雌胚胎干细胞集落的建立

ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＳＴＥＭＣＥＬＬＣＯＬＯＮＩＥＳＦＲＯＭＰＡＲＴＨＥＮＯＧＥＮＥＴＩＣＡＬＬＹＤＥＲＩＶＥＤＢＬＡＳＴＯＣＹＳＴＳＯＦＭＯＵＳＥ小鼠孤雌胚胎干细胞集落的建立ＫｅｙｗｏｒｄｓＭｏｕｓｅ,Ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃｅｍ...     

Regeneration of inner ear cells from stem cell precursors--a future concept of hearing rehabilitation?

The use of stem cells offers new and powerful strategies for future tissue development and engineering. Common features of stem cells are both their capacity for self-renewal and the ability to differentiate into mature effector cells. Since the establishment of embryonic stem cells from early human embryos, research on and clinical application of human ES cells belong to the most controversial topics in our society. Great hopes are based upon the remarkable observation that human ES cells can be greatly expanded in vitro, and that they can differentiate into various clinically important cell types. Recent advances in the cloning of mammals by nuclear transplantation provide new concepts for autologous replacement of damaged and degenerated tissues. In contrast, somatic stem cells of the adult organism were considered to be more restricted in their developmental potential. However, recent investigations suggest that somatic stem cells may have a wider differentiation potential than previously thought. In otology, initial experiments have revealed neural stem cell survival in cochlear cell cultures and under neurotrophin influence, neural stem cells seemed to develop into a neuronal phenotype. Further studies have to be carried out to investigate the full potential of stem cells as well as the molecular mechanisms that are involved in regulating cellular identity and plasticity. Clinically, advances in stem cell biology may provide a permanent source of replacement cells for treating human diseases and could open the development of new concepts for cell and tissue regeneration for a causal treatment of chronic degenerative diseases.     

Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.     

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.     

新物种诱导多能干细胞的研究进展

胚胎干细胞（embryonic stem cells,ESC）在人类遗传病学研究、疾病模型建立、器官再生以及动物物种改良和定向变异等方面的地位是其他类型的细胞不可取代的。但是,由于实验技术和体外培养条件的限制,除了小鼠、恒河猴和人之外,大鼠、猪、牛、羊等其他哺乳动物的ES细胞系被证明很难获得。先后有多个研究小组报道了他们利用新兴的诱导多能干细胞（induced pluripotent stem cells,iPS细胞）技术成功建立大鼠和猪的iPS细胞系的研究成果。迄今为止,这两个物种是在未成功建立ES细胞系之前利用iPS技术建立多能干细胞系的成功范例。这些研究对于那些还未建立ES细胞的物种建立多能干细胞系提供了一种新的方案,也将给这些物种的胚胎干细胞的建立、基因修饰动物的产生以及人类医疗事业的促进和发展带来新的希望。     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Embryonic stem cells

Embryonic stem cells have huge potential in the field of tissue engineering and regenerative medicine as they hold the capacity to produce every type of cell and tissue in the body. In theory, the treatment of human disease could be revolutionized by the ability to generate any cell, tissue, or even organ, 'on demand' in the laboratory. This work reviews the history of murine and human ES cell lines, including practical and ethical aspects of ES cell isolation from pre-implantation embryos, maintenance of undifferentiated ES cell lines in the cell culture environment, and differentiation of ES cells in vitro and in vivo into mature somatic cell types. Finally, we discuss advances towards the clinical application of ES cell technology, and some of the obstacles which must be overcome before large scale clinical trials can be considered.     

Principles of ethical decision making regarding embryonic stem cell research in Germany

The availability of embryonic stem (ES) cells isolated from human blastocysts may open novel avenues for medical treatment of otherwise incurable diseases. Yet the generation of human ES cells requires the destruction of early human embryos. This confronts us with the moral problem of whether it is justifiable to sacrifice human life in order to treat other human life. This article outlines the development of the German debate about research with ES cells and explicates the arguments that are central to that debate with respect to the aims and means of research with ES cells. With regard to the means, the isolation of ES cells from human embryos raises the question of the moral status of the human embryo. A restrictive position acknowledges the human dignity of the embryo in its very early stage of development and claims that the embryo's life must be protected accordingly. In contrast, a gradualist position acknowledges human dignity, and therefore the full level of protection, only when the embryo has reached a certain stage of development. In addition, the intentions behind the generation of human embryos, i.e. exclusively for research purposes, and the mode of generating them, i.e. by nuclear transfer technology, have strong ethical relevance in the German debate. Based on these results, the ethical reasoning underlying the draft of a Stem Cell Act recently passed by the German Parliament is outlined.     

Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.

Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.     

Trichostatin A induces myocardial differentiation of monkey ES cells

Human embryonic stem (ES) cell lines are one of the possible sources of cardiac myocytes to be transplanted in patients with end-staged heart failure. However, prior to the application of human of ES cells for heart failure therapy, it is critical to validate their clinical use in large animals such as primates. Cynomolgus monkey ES cells have similar properties to human ES cells and can be used for primate studies. We demonstrate that 24-h stimulation by a histone deacetylase inhibitor, trichostatin A (TSA) facilitated myocardial differentiation of monkey ES cells with embryonic bodies that were seeded on gelatin-coated dishes. TSA-induced acetylating of histone-3/4 and expression of p300, one of the intrinsic histone acetyltransferases. Thus, such induction as well as inhibition of histone deacetylase may be involved in TSA-induced differentiation of cynomolgus monkey ES cells into cardiomyocytes.     

Increasing the cell number of host tetraploid embryos can improve the production of mice derived from embryonic stem cells

Tetraploid (4n) embryo complementation assay has shown that embryonic stem (ES) cells alone are capable of supporting embryonic development (ES mouse), allowing the recovery of mouse lines directly from cultured ES cell lines. Although the advantages of this technique are well recognized, it remains inefficient for generating ES mice. In the present study, we investigated the effects of cell number of host 4n embryos on the production of ES mice. Four independent ES cell lines (two general ES cell lines and two nuclear transfer-derived ES cell lines) were used, and each cell line was aggregated with single (1x) to triple (3x) host 4n embryos. We found that birth rate of ES mice using 1x 4n embryos was quite low (0-2%) regardless of cell line, whereas except for one cell line, approximately 6-14% of embryos developed to full term in the case of 3x 4n embryos. Contamination of host 4n cells in ES mice was quite rare, being comparable to that generated using general methods even if they were delivered from 3x 4n host embryos. These results demonstrate that the use of 3x 4n embryos is effective for generating ES mice. Our technique described here will be applicable to any ES cell line, including general ES cell lines used for gene targeting.     

Generating Embryonic Stem Cells from the Inbred Mouse Strain DBA/2J,a Model of Glaucoma and Other Complex Diseases

Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background.     

Embryonic stem cells in non-human primates: An overview of neural differentiation potential

Non-human primate (NHP) embryonic stem (ES) cells show unlimited proliferative capacities and a great potential to generate multiple cell lineages. These properties make them an ideal resource both for investigating early developmental processes and for assessing their therapeutic potential in numerous models of degenerative diseases. They share the same markers and the same properties with human ES cells, and thus provide an invaluable transitional model that can be used to address the safety issues related to the clinical use of human ES cells. Here, we review the available information on the derivation and the specific features of monkey ES cells. We comment on the capacity of primate ES cells to differentiate into neural lineages and the current protocols to generate self-renewing neural stem cells. We also highlight the signalling pathways involved in the maintenance of these neural cell types. Finally, we discuss the potential of monkey ES cells for neuronal differentiation.