Contrasting human and mouse embryonic stem cell self‐renewal using Bayesian network machine learning

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.

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

Embryonic stem (ES) cells are derived from the epiblast of the inner cell mass (ICM) of a blastocyst. These are pluripotent cells and can give rise to all derivatives of the three primary germ layers ectoderm, endoderm, and mesoderm. ES cells require specific signals for specific lineage of differentiation if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma (Wu et al., 2007). The techniques for culturing mouse embryonic stem (ES) cells from the inner cell mass of the preimplantation blastocyst were first done in 1981 (Martin, 1981), and versions of these standard procedures are used today in laboratories throughout the world. The first successful derivation of human ES (hES) cells was reported by Thomson et al. (Thomson et al., 1998). They isolated and plated the cells onto mitotically inactivated MEF (mouse embryonic fibroblast) cells. In 2000, Reubinoff et al. confirmed that hES cells could be efficiently derived from surplus embryos and possess the differentiation potential under in vitro conditions. Since then, there has been rapid progress made and numerous studies have described the derivation of new hES cell lines including methods of growing both undifferentiated hES cells and their differentiated progeny. In last 6 years, there has been exponential increase in methods to improve culture conditions, differentiation patterns to produce human cells for transplantation and drug testing (Trounson, 2006, Gepstein, 2002) and genetic manipulation (Draper et al., 2004, Zwaka and Thomson, 2003). Generating cultures of mouse or human ES cells that remain in a proliferating and undifferentiated state is multistep process. Typically, the inner cell mass of a preimplantation blastocyst is removed from the trophectoderm that surrounds it and cultured in the small plastic culture dishes containing growth medium supplemented with fetal calf serum. The culture dishes are sometimes coated with a layer of nondividing cells, which are often MEF cells that have been chemically inactivated so they will not divide. Mouse ES cells can be grown in vitro without feeder layers if the cytokine leukemia inhibitory factor (LIF) is added to the culture medium, but human ES cells do not respond to LIF. The process of generating an embryonic stem cell line is somewhat difficult, so lines are not produced every time from the preimplantation-stage embryo maintained in a culture dish. However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed, and plated into several fresh culture dishes. The process of re-plating or

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.

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.

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.

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

The nucleosome remodeling and deacetylation (NuRD) complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered a key player in the formation of a functional NuRD complex. The recruitment of MBD3 to methylated promoters may require other prerequisite factors. We show that cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), an essential gene for early embryonic development, plays a role in pluripotency of ESC by engaging MBD3 to the promoter region of Wnt signaling genes. The occupancy of MBD3 on several promoters of Wnt genes was significantly lost in the absence of CDK2AP1, resulting in hyperactivation of Wnt. We propose that the transcriptional modulation of the Wnt pathway mediated by NuRD requires the presence of essential auxiliary components such as CDK2AP1, which may aid the association of the complex with specific focal regions of the target promoters.

Derivation, propagation and differentiation of human embryonic stem cells

Recent success with immunosuppression following islet cell transplantation offers hope that a cell transplantation treatment for type 1 (juvenile) diabetes may be possible if sufficient quantities of safe and effective cells can be produced. For the treatment of type 1 diabetes, the two therapeutically essential functions are the ability to monitor blood glucose levels and the production of corresponding and sufficient levels of mature insulin to maintain glycemic control. Stem cells can replicate themselves and produce cells that take on more specialized functions. If a source of stem cells capable of yielding glucose-responsive insulin-producing (GRIP) cells can be identified, then transplantation-based treatment for type 1 diabetes may become widely available. Currently, stem cells from embryonic and adult sources are being investigated for their ability to proliferate and differentiate into cells with GRIP function. Human embryonic pluripotent stem cells, commonly referred to as embryonic stem (ES) cells and embryonic germ (EG) cells, have received significant attention owing to their broad capacity to differentiate and ability to proliferate well in culture. Their application to diabetes research is of particular promise, as it has been demonstrated that mouse ES cells are capable of producing cells able to normalize glucose levels of diabetic mice, and human ES cells can differentiate into cells capable of insulin production. Cells with GRIP function have also been derived from stem cells residing in adult organisms, here referred to as endogenous stem cell sources. Independent of source, stem cells capable of producing cells with GRIP function may provide a widely available cell transplantation treatment for type 1 diabetes.

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.

Previously we reported that the spindle assembly checkpoint (SAC), which is coupled in somatic cells, is uncoupled from apoptosis-initiation in mouse and human ESCs. This condition allows ESCs to tolerate and proliferate as polyploidy/aneuploid cells. Proper function of the SAC is vital to prevent polyploidy/aneuploidy during ex-vivo HSC expansion. Here we address, for the first time, whether hematopoietic stem cells are more like ESC or somatic cells with respect to SAC-apoptosis coupling. Using multiparametric permeablized cell flow-cytometric analysis to identify and analyze the mouse sca 1+/c-kit+/lin− (LSK) population, we found the mitotic spindle checkpoint to be functional in primary murine LSK cells, a population enriched in primitive hematopoietic stem/progenitor cells, after prolonged activation of the SAC by microtubule-depolymerizing agents such as nocodazole. HSCs can efficiently initiate apoptosis after activation of the SAC in LSK cells as indicated by increased hypopdiploidy and increased levels of activated caspase-3 suggesting that HSCs behave more like somatic cells instead of ESCs with respect to this important cell cycle checkpoint. We conclude that mouse HSCs are not subject to the same kinds of chromosomal-instability as are ESCs, knowledge that might aid in optimizing in-vitro culture and expansion of human bone marrow or cord blood HSC for clinical applications.

Cell transplantation is being extensively explored as a means of treating many human degenerative diseases. The leukodystrophies are examples of neurological disorders where new therapeutic strategies, either cellular or molecular, will be required to repair the central nervous system (CNS) of affected patients. Much hope is being pinned on the use of human embryonic stem (ES) cells as the exogenous source of neurons and glia to replace dysfunctional or dying cells in the CNS. In the case of leukoencephalopathies, the goal is to generate oligodendrocytes or other myelinating cells such as Schwann cells from ES cells, to myelinate or remyelinate CNS axons on transplantation. Experimental data suggests that mouse ES cells have this capacity, but at present differentiation of oligodendrocytes in sufficient numbers from human ES cells is not possible. It may in fact be more feasible to isolate oligodendrocytes from human neural stem cells derived from the fetal brain, but the source of these is in short supply and, like that of ES cells, is ethically controversial. None the less, it appears certain that either of these two sources will eventually give rise to sufficient numbers of neural stem cells or oligodendrocyte progenitors that have greater capacity for repair than such cells derived from the adult brain. Once the primary technical issues concerning human ES cell differentiation have been overcome, the most likely first clinical target will be Pelizaeus-Merzbacher disease. However, widespread dissemination of cells throughout the CNS may be required for functional improvement; hence diseases such as adrenoleukodystrophy may also be considered as therapeutic targets.

Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4, Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen, Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but, until now, its role in mammalian development and ESCs has not been explored. In this study, we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs, we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly, knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2, Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore, we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle.