Nuclear Reprogramming of Somatic Nucleus Hybridized With Embryonic Stem Cells by Electrofusion

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

The twisted path to pluripotency.

Germline development from human pluripotent stem cells toward disease modeling of infertility

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Embryonic stem (ES) cells have generated enormous interest because of their capacity to self-renew and differentiate into various cell types in vitro. Although numerous problems are encountered in the use of ES cells for regenerative medicine, such as ethical issues associated with the use of stem cells established from terminated human embryos and immunorejection due to transplantation of allogenic ES cell-derived cells into patients, recent technologies to generate induced pluripotent stem (iPS) cells from adult somatic cells have provided alternative ways to access pluripotent stem cells (Takahashi et al., 2007). However, the practical application of these pluripotent stem cells has yet to emerge, and regulatory mechanisms are not well known. Moreover, precise differentiation methodologies of ES and iPS cells have not been developed. These problems cause difficulties in the manipulation of pluripotent stem cells and derivation of functionally differentiated cells. Detailed analysis of the transcriptome has allowed elucidation of transcription networks that regulate the pluripotency of these stem cells. However, the specific nuclear infrastructures that maintain the pluripotent stem cell-specific transcription network have not yet been elucidated. We used proteomics to analyze the nuclear protein machinery in stem cells and identified some crucial components for the maintenance of pluripotent stem cells. In addition, various growth factors and extracellular matrix components regulate the pluripotency and differentiation of stem cells. Therefore, the cell surface receptors that bind these regulatory factors are important for the precise regulation of stem cells. We have also explored stem cell-specific cellsurface markers by proteomic analysis of mouse ES cells. These cell-surface membrane proteins can be useful to manipulate pluripotent stem cells. In this chapter, we describe some examples of new findings elucidated by proteomic analysis of ES cells.

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Embryonic, fetal, and adult stem cells have been extensively tested in the past decade as candidates for cell replacement therapy of severe heart failure. Among them, embryonic stem (ES) cells have been regarded as an especially valuable source of therapeutic cells due to their unlimited growth in culture and well-established cardiogenic potential in vitro. ES cell preparations that were tested in animal models of heart infarction ranged from fully undifferentiated and cardiac-committed ES cells to partially or highly enriched ES cell-derived cardiomyocytes. Here we critically review the current literature on use of fully undifferentiated ES cells for cardiac repair, elaborate on the tumorigenic risk of ES cells and pluripotent cells in general, and summarize strategies for elimination of this threat as an important step toward translation of ES cell-based therapies to clinic. This discussion is also highly relevant for clinical applicability of newly developed autologous ES cell-like stem cells, so-called induced pluripotent stem cells, which circumvent ethical and, to some extent, immunological concerns linked to the use of blastocyst-derived ES cells, but still possess high tumorigenic potential.

Human Embryonic Stem Cells . Edited by J. Odorico, S. Zhang and R. Pedersen . (Pp. xxii + 391 , illustrated , ISBN 18599 62785 , £80 hardback.) Abingdon, UK : BIOS Scientific/Garland Science . It is now nearly seven years since the first description of human embryonic stem (ES) cell lines was published by Jamie Thomson. That event, closely apposed to the reports of the cloning of sheep (‘Dolly’), and then mice, by somatic nuclear transfer, catalysed substantial interest in the idea of regenerative medicine and the possibility that stem cells of all sorts might be harnessed for the replacement of tissues lost to accident or disease. At about the same time, a number of papers appeared, reporting that various somatic stem cells – which were previously thought to be committed to lineages from within their tissue of origin – might indeed exhibit substantial plasticity and be able to generate terminally differentiated cells corresponding to a much wider range of cell types. Thus, the current era of stem cell biology was born, though the concept of stem cells and their application in medicine is much older. One consequence of this resurgent interest in stem cell biology has been the publishing of several multi-author volumes devoted to stem cells and their potential. The volume Human Embryonic Stem Cells, edited by J. Odorico, S. Zhang and R. Pedersen, is the latest in a series produced by different publishing houses over the past few years. Paradoxically, however, as Jamie Thomson points out in his foreword to the present volume, progress in the field has been slow over this time. Prospects for adult stem cells have been dimmed by controversy over the phenomenon of ‘plasticity’, with the suggestion that at least some of the reported cases might be artefactual, depending upon cell fusion or rare transdifferentiation events. At the same time, progress with human ES cells was initially hindered by the difficulty of access to established lines, and the complexity of maintaining and expanding the cultures when they could be obtained. Although mouse ES cells have been available for over 20 years, they have mostly been used as tools to produce transgenic mice. With notable exceptions, few have investigated their cell biology for its own sake, and so there has been relatively little experience from studies of mouse ES cells to guide the study of their human counterparts. The development of ES cell lines was a systematic progression from the study of teratocarcinomas in the 1970s, by those who thought that these tumours might provide key insights into the mechanisms of embryonic development. It is then perhaps ironic that clues about how to control the behaviour of human ES cells in culture are now being provided by our detailed knowledge of developmental genetics, especially of the mouse. However, the human ES cell field is changing. Many laboratories in many countries have now derived human ES cell lines. In the current International Stem Cell Initiative, a collaborative venture to compare the properties of human ES cell lines derived worldwide, 75 independent lines derived in 17 laboratories and ten countries have been enrolled in the study. Meanwhile, reports are now beginning to appear in prominent journals, addressing the molecular mechanisms of human ES cell proliferation and differentiation – not merely describing their characteristics and potential for diverse differentiation. The present volume, unlike many of its competitors, focuses explicitly on human ES cells, and may be thought to mark the end of the first phase of human ES cell research. It brings together a series of authors who have contributed significantly to the field and it balances chapters discussing the basic biology of ES cells with others discussing their differentiation along specific lineages and eventual application. Despite the focus on ES cells, two early chapters provide useful comparative reviews of recent studies of adult stem cell plasticity and of mesenchymal stem cells. Further chapters address potential issues that will need to be addressed as derivatives of human ES cells are developed for eventual clinical application. Included here are two interesting and useful reviews of the ethical, legal and intellectual property aspects of human ES cell research and application. Undoubtedly, research in the new field of human ES cell biology is beginning to advance rapidly, so that it might be considered that the present volume will become rapidly obsolete. However, its well-organized and well-written chapters provide a valuable reference to the current state of the field for newcomers and established human ES cell researchers alike. Although our understanding of molecular mechanisms and the means of manipulating human ES cells is likely to change significantly in the future, much of what is contained in this volume will remain fundamental, and so of value to those working in this exciting new area of research for quite a few years to come.

Pluripotency in the early embryo is defined as the capacity of a single cell to generate all lineages of the adult organism. In vivo this property is possessed only transiently by the cells of the epiblast, but it can be indefinitely “captured” in vitro by deriving embryonic stem (ES) cells from the inner cell mass of the blastocyst. A second property that characterizes ES cells is self-renewal, the capacity to generate more stem cells. Self-renewal requires the coordination of cell proliferation and cell-fate choice (Orford and Scadden, 2008). Originally, mouse ES cells were expanded on mitotically inactivated fibroblasts, in a culture medium containing fetal bovine serum and the leukemia inhibitory factor (LIF). Under these conditions ES cells are metastable, since they show heterogeneity both in morphology and expression of the core pluripotency factors Oct4 and Nanog due to differentiation signals coming from the serum components (Nichols and Smith, 2009). In serum + LIF medium, ES cells depend on the transcription factor and proto‐oncogene Myc for pluripotency and self‐renewal (Smith et al., 2010). Myc proteins have been shown to repress the expression of the primitive endoderm master regulator Gata6, and control the cell cycle by regulating the mir-17-92 miRNA cluster. The laboratory of Austin Smith has recently shown that the culture in the absence of serum but in the presence of two inhibitors (2i + LIF) of the Erk and glycogen synthase kinase-3 (GSK3) pathways is sufficient to stabilize ES cells in a more naïve, so-called ground state of pluripotency, which more closely resembles the status of the inner cell mass of the blastocyst (Ying et al., 2008). Since different culture environments impose distinctive transcriptional and epigenetic properties on mouse ES cells (Marks et al., 2012), we re-evaluated the expression and requirement of the Myc proteins in the naïve state of pluripotency. Making use of ES cells expressing a Myc-GFP reporter from the endogenous c-myc locus, we found that c-Myc protein expression is significantly lower in naïve ES cells compared to cells cultured in serum + LIF. When both c- and N-myc genes are conditionally deleted (Myc dKO) using the Cre-loxP system, naïve mouse ES cells undergo cell cycle arrest. Although this type of cell cycle behavior with a decreased rate of cell division is often associated with differentiation, Myc dKO ES cells form smaller but undifferentiated colonies. Most surprisingly, Myc dKO ES cells maintain the expression of the core stem cell factor network including Oct4, Nanog and Sox2 both at the RNA and the protein level. To determine the molecular properties of Myc dKO ES cells, we performed RNA‐seq analysis 24 and 96 hours after Cre induction. Our results show that Myc is not exclusively regulating the cell cycle machinery but is directly and indirectly involved in multiple aspects of ES cell metabolism. In the absence of both Myc proteins, expression of genes controlling and executing ribosomal biogenesis as well as protein and DNA synthesis is strongly down-regulated, leading to a state of “metabolic dormancy”. The state of “metabolic dormancy” of ES cells in vitro resembled the state of embryonic diapause in vivo. Diapause is a poorly understood phenomenon of reversible arrest of embryonic development prior to implantation. In mice, facultative diapause occurs to delay implantation of newly formed embryos when the mother is feeding a previous litter. When we compared our data to reported global gene expression profiling of diapause embryos (Hamatani et al., 2004), we observed that dKO Myc ES cells possess surprising similarity with the dormant blastocyst, characterized by reduced DNA synthesis and cell division, low metabolic activity and activation of the insulin pathway (Given, 1988; Hamatani et al., 2004). These data suggest that, in the absence of differentiation cues, c-Myc and N-Myc control the cell cycle of ES cells and the transcriptional networks responsible for the entire metabolism and biosynthesis pathways while the pluripotency network is controlled by other means. Loss of Myc activity has dramatic consequences on the metabolic status of ES cells promoting their entry into a state of likely reversible dormancy closely resembling arrested diapause embryos.

Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.

Recipes for cooking with stem cells

A wealth of evidence attests that the organs of developing embryos, particularly the developing brain, are acutely sensitive to chemical perturbations. However, scientists know very little about how exposures to specific endogenous chemicals actually impact human development or children’s ability to learn. And there are almost no data on how the vast majority of the 84,000 chemicals currently listed in the Toxic Substances Control Act (TSCA) Inventory1—including most of the 201 compounds known to be neurotoxic to adults and the 1,000 chemicals shown to be neurotoxic to animals2—may affect developing infants. It is also unclear whether testing with animals always provides accurate insights into human developmental susceptibility. A new line of research based on human stem cells is providing important insights into how chemicals may affect neonatal development. Stem cells are the master cells capable of producing some or all of the 200-plus different types of cells in the human body. In time, some researchers believe stem cells may enable scientists to amass far more data on how exposure to environmental chemicals affects human development, particularly the development of the brain. Now is a “critical time to be talking about stem cell research in the environmental health context,” says Tracey Woodruff, director of the Program on Reproductive Health and the Environment at the University of California, San Francisco (UCSF) Medical School.

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%-40% of each cell type differentiated into Flk1(+) cells. The sorted Flk1(+) cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1(+) mGS cells generated more contractile colonies than did the Flk1(+) ES cells, whereas the Flk1(+) EG cells generated equivalent numbers as the Flk1(+) mGS cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of differentiation markers such as Rex1, FGF-5, GATA-4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0-4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT-PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1(+) mGS cells express mature cardiac cell-specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.