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PubMed2001

[Progress in Research on Embryonic Stem Cells]

Embryonic stem cells are continuously growing stem cell lines of embryonic origin. The distinguishing features of ES cells are their capacity to be maintained in an undifferentiated state indefinitely in culture and their potential to develop into various kinds of cells in the body. At molecular level, in vivo and in vitro differentiation of ES cells are similar in many ways. To date, ES cells can differentiate in vitro into myocytes and myocardial cells, neural precursor cells, hematopoietic cells and endothelial cells. Recent evidence suggests that hematopoietic stem cells (HSCs) originate from a region within the embryo proper. It is likely that the genesis of HSCs is regulated by embryonic growth factors, which have not been discovered yet. ES cells have provided a convenient and effective experimental approach to define early hematopoietic process within embryos. Research on ES cells has broad application prospect.

Journal Article↗

PubMed2006

Murine embryonic stem cells.

Embryonic stem (ES) cells are derived from preimplantation stage mouse embryos at the time when they have reached the blastocyst stage. It is at this point that the first steps of differentiation take place during mammalian embryonic development. The individual blastomeres now start to organize themselves into three distinct locations, each encompassing a different cell type: outside epithelial cells, trophectoderm; cells at the blastocele surface of the inner cell mass (ICM), the primitive endoderm; and inside cells of the ICM, the primitive ectoderm. ES cells originate from the third population, the primitive ectoderm, which is a transiently existing group of cells in the embryo. Primitive ectoderm cells diminish within a day as the embryo is entering into the next steps of differentiation. ES cells, however, while retaining the property of their origin in terms of developmental potential, also have the ability to self-renew. It is hence important to realize that ES cells do not exist in vivo; they should be regarded simply as tissue culture artifact. Nevertheless, these powerful cells have the potential to differentiate into all the cells of the embryo proper and postnatal animal. Furthermore, they retain the limitation of their origin through their inability to contribute to the trophectoderm lineage (the trophoblast of the placenta) and the lineages of the primitive endoderm, the visceral and parietal endoderm. Due to these unique features, we must admit that even if we regard ES cells as products of in vitro culture and should not compare them to true somatic stem cells found in the adult organism, they certainly offer us a fantastic tool for genetic, developmental, and disease studies.

Animals↗

PubMed2004

Progress with nonhuman primate embryonic stem cells.

Embryonic stem cells hold potential in the fields of regenerative medicine, developmental biology, tissue regeneration, disease pathogenicity, and drug discovery. Embryonic stem (ES) cell lines are now available in primates, including man, rhesus, and cynomologous monkeys. Monkey ES cells serve as invaluable clinically relevant models for studies that can't be conducted in humans because of practical or ethical limitations, or in rodents because of differences in physiology and anatomy. Here, we review the current status of nonhuman primate research with ES cells, beginning with a description of their isolation, characterization, and availability. Substantial limitations still plague the use of primate ES cells, such as their required growth on feeder layers, poor cloning efficiency, and restricted availability. The ability to produce homogenous populations of both undifferentiated as well as differentiated phenotypes is an important challenge, and genetic approaches to achieving these objectives are discussed. Finally, safety, efficiency, and feasibility issues relating to the transplantation of ES-derived cells are considered.

Animals↗

PubMed2007

Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells.

Embryonic stem cells (ES cells) are derived from inner cell mass (ICM). The self-renewal and pluripotency are the main specificities of ES cells, which are likely to reveal a deeper understanding of human cellular biology and which are considered to be promising sources for cell therapy to treat patients with degenerative diseases in clinical. Growth of ES cells as a pluripotent population requires a balance between survival, proliferation, and self-renewal signals. In fact, the precise mechanism that regulates stem cell self-renewal and pluripotency remains largely unknown. Recently, in vitro and in vivo studies have identified several genetic regulators that may play important roles in the self-renewal and pluripotency process of human and mouse ES cells, including extracellular signaling factors, transcription factors, cell-cycle regulators, microRNA, genes implicated in chromosomal stability, and DNA methylation. In this review, we will summarize the currently known molecular regulators for ES cells self-renewal, and we will propose some possibilities to explain the ways in which these distinct pathways might interact.

Animals↗

PubMed2006

Transcriptional profiling of rhesus monkey embryonic stem cells.

Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

Animals↗

PubMed2006

Methylation status of imprinting centers for H19/IGF2 and SNURF/SNRPN in primate embryonic stem cells.

Embryonic stem cells (ESCs) hold promise for cell and tissue replacement approaches to treating human diseases based on their capacity to differentiate into a wide variety of somatic cells and tissues. However, long-term in vitro culture and manipulations of ESCs may adversely affect their epigenetic integrity, including imprinting. We have recently reported aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ESC lines, whereas SNRPN and NDN were normally imprinted and expressed predominantly from the paternal allele. The dysregulation of IGF2 and H19 that is associated with tumorigenesis in humans may result from improper maintenance of allele-specific methylation patterns at an imprinting center (IC) upstream of H19. To test this possibility, we performed methylation analysis of several monkey ESC lines by genomic bisulfite sequencing. We investigated methylation profiles of CpG islands within the IGF2/H19 IC harboring the CTCF-6 binding site. In addition, the methylation status of the IC within the promoter/exon 1 of SNURF/SNRPN known as the Prader-Willi syndrome IC was examined. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analyzed ESC lines, whereas the SNURF/SNRPN IC was differentially methylated, consistent with monoallelic expression.

Animals↗

PubMed2006

Formation of gut-like structures in vitro from mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into all cell types originating from the three germ layers; however, there are still few reports about the formation of functional organs from embryonic stem cells. Recently, we reported that by hanging drops of mouse ES cells, embryoid bodies (EBs) formed gut-like structures in vitro composed of three layers corresponding to the epithelium, lamina propria, and musculature. The morphological features and the process of formation are similar to gut and its organogenesis in vivo. Thus, this is a good model for development of the gut and a useful tool for analysis of the factors required for gut organogenesis. The protocol basically involves a method of hanging drops to make EBs, which are then plated on coated dishes for outgrowth. EBs develop to form gut-like structures when induced to spontaneously enter a program of differentiation in vitro without addition of any extrinsic factors.

Animals↗

PubMed2006

A protein interaction network for pluripotency of embryonic stem cells.

Embryonic stem (ES) cells are pluripotent and of therapeutic potential in regenerative medicine. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog, which is central to the maintenance of ES cell pluripotency. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency.

Animals↗

PubMed2003

Generation of structures formed by lens and retinal cells differentiating from embryonic stem cells.

Embryonic stem cells have the potential to give rise to all cell lineages when introduced into the early embryo. They also give rise to a limited number of different cell types in vitro in specialized culture systems. In this study, we established a culture system in which a structure consisting of lens, neural retina, and pigmented retina was efficiently induced from embryonic stem cells. Refractile cell masses containing lens and neural retina were surrounded by retinal pigment epithelium layers and, thus, designated as eye-like structures. Developmental processes required for eye development appear to proceed in this culture system, because the formation of the eye-like structures depended on the expression of Pax6, a key transcription factor for eye development. The present culture system opens up the possibility of examining early stages of eye development and also of producing cells for use in cellular therapy for various diseases of the eye.

Animals↗

PubMed2006

Pluripotency: capacity for in vitro differentiation of undifferentiated embryonic stem cells.

Embryonic stem (ES) cells, the pluripotent cells of early embryos have been successfully cultured as undifferentiated cells. The cells are characterized by two unique properties, unlimited self-renewal capacity and the ability to differentiate into all cells of the body. Because of the high in vitro differentiation potential, ES cells have been used as model system in cell and developmental biology. Here we present methods that use mouse embryonic stem cells for the in vitro differentiation and characterization of neuronal, cardiac, pancreatic and hepatic cells, derivatives of the ectoderm, mesoderm and endoderm, respectively. In the future, differentiated cells may be also generated from human ES cells by cultivation of early embryos or from reprogrammed cells derived by nuclear transfer. Such cells could represent potential sources for tissue repair of serious human diseases.

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PubMed2004

Self-renewal vs. differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.

Animals↗

PubMed2003

Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells.

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after approximately 4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.

Adrenergic beta-Agonists↗

PubMed2001

Potential of embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.

Animals↗

PubMed2005

Pharmaceutical applications of embryonic stem cells.

Embryonic stem cells (ESCs) will become a source of all adult differentiated cells once reliable protocols for directed differentiation have been established. This resource will revolutionise laboratory cell biology and will provide much improved cell culture models for discovery and development of drugs, and fundamental studies of the genetic basis of disease. These are early days, and there are few examples of practical outcomes in the pharmaceutical world, though in recent years some drug-like molecules, which direct differentiation, have been discovered. At this stage the focus is on understanding the signalling systems and transcription factors that drive differentiation, on characterisation and isolation of precursor cells, and on establishing methods to improve the homogeneity of differentiated cells derived from ESCs. There is particular interest in establishing methods for deriving fully differentiated human cells from human ESCs (HESCs), but there are challenges to be met before HESC technology can be taken up in a widespread manner. Protocols for expansion of HESC cultures are labour-intensive at present. There is an urgent need for discovery of factors that will allow serum-free (and feeder cell-free) expansion of HESCs, and which can be applied to the majority of HESC lines. Our expectation is that adequate protocols will emerge in the coming years opening the way for development of many cell culture tools in the pharmaceutical industry. Here we review the current status and future prospects for this exciting field and encourage pharmaceutical scientists to play a role in directing its development.

Animals↗

PubMed2006

Mechanisms of self-renewal in human embryonic stem cells.

Embryonic stem cells (ESCs) are the pluripotent cell population derived from the inner cell mass of pre-implantation embryos and are characterised by prolonged self-renewal and the potential to differentiate into cells representing all three germ layers both in vitro and in vivo. Preservation of the undifferentiated status of the ESC population requires the maintenance of self-renewal whilst inhibiting differentiation and regulating senescence and apoptosis. In this review, we discuss the intrinsic and extrinsic factors associated with self-renewal process, together with possible signalling pathway interactions and mechanisms of regulation.

Biomarkers↗

PubMed2006

Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells.

Embryonic stem (ES) cells can self-renew indefinitely without losing their differentiation ability to any cell types. Phosphoinositide-3 kinase (PI3K)/Akt signaling plays a pivotal role in various stem cell systems, including the formation of embryonic germ (EG) cells from primordial germ cells and self-renewal of neural stem cells. Here, we show that myristoylated, active form of Akt (myr-Akt) maintained the undifferentiated phenotypes in mouse ES cells without the addition of leukemia inhibitory factor (LIF). The effects of myr-Akt were reversible, because LIF dependence and pluripotent differentiation activity were restored by the deletion of myr-Akt. In addition, myr-Akt-Mer fusion protein, whose enzymatic activity is controlled by 4-hydroxy-tamoxifen, also maintained the pluripotency of not only mouse but also cynomolgus monkey ES cells. These results clearly demonstrate that Akt signaling sufficiently maintains pluripotency in mouse and primate ES cells, and support the notion that PI3K/Akt signaling axis regulates 'stemness' in a broad spectrum of stem cell systems.

Animals↗

PubMed2005

Mixl1 and oct4 proteins are transiently co-expressed in differentiating mouse and human embryonic stem cells.

Embryonic stem cells (ESCs) have the capacity to form all the tissues in the body and hence, directed differentiation of ESCs along specific lineages represents a means to generate therapeutically useful cell types. It has been postulated that, during in vitro differentiation, ES cells sequentially pass through similar developmental stages as cells in the embryo. The availability of reagents that identify these stages would facilitate the monitoring and optimization of ESC differentiation. One key stage, the development of endodermal and mesodermal precursors in the early embryo, is marked by the transient expression of the transcription factor, Mixl1 and the stem cell gene, Oct4. In order to identify corresponding cells during ESC differentiation, we generated monoclonal antibodies to the Mixl1 protein that robustly detected both mouse and human proteins. Intracellular flow cytometry was used to show that approximately 90% of differentiating mouse ESCs transiently co-expressed Oct4 and Mixl1 proteins and that a subset of differentiating human ES cells also coexpressed MIXL1 and OCT4 proteins. These experiments have demonstrated for the first time by protein expression that both human and mouse ESCs passed through developmental stages during in vitro differentiation that corresponded to those observed in early mammalian development. Furthermore, these studies confirmed that anti-Mixl1 antibodies are a valuable reagent for monitoring ESC differentiation and will facilitate the efficient generation of clinically relevant cell types.

Animals↗

PubMed2004

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.

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