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During implantation, the murine embryo transitions from a “quiet” into an active metabolic/proliferative state, which kick‐starts the growth and morphogenesis of the post‐implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine‐tunes the expression of genes that encode ribosomal proteins and is required for proper tissue‐scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage’s high‐energy demands for cell proliferation and morphogenesis., Ronin enables active energy production, which is required for the transition from metabolically “quiet” (pre‐implantation) or “quiescent” (diapause) states into a proliferative (post‐implantation) state of embryo development.

Hurting the virus by targeting the host Many host proteins play a role in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and some are required for viral replication and translation. There are efforts toward finding drugs that target viral proteins, but a complementary approach is to target these required host proteins. White et al. explored the antiviral activity of the cyclic depsipeptide drug plitidepsin, which targets the hosts cell's translational machinery (see the Perspective by Wong and Damania). The authors show that in cells, the drug is substantially more potent than remdesivir against SARS-CoV-2, with limited cellular toxicity. Prophylactic treatment protected mice against SARS-CoV-2 infection, so further investigation of plitidepsin as a therapeutic is warranted. Science, this issue p. 926; see also p. 884, Plitidepsin is a host-targeted antiviral against SARS-CoV-2, with potent efficacy both in vitro and in mouse models., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

Spinocerebellar ataxias (SCAs) are a group of genetically heterogeneous inherited neurodegenerative disorders characterized by progressive ataxia and cerebellar degeneration. Here, we tested if Ronin (Thap11), a polyglutamine-containing protein encoded in a region on human chromosome 16q22.1 that has been genetically linked to SCA4, can be connected with SCA disease in a mouse model. We report that transgenic expression of Ronin in mouse cerebellar Purkinje cells leads not only histopathologically to detrimental loss of Purkinje cells but also phenotypically to the development of severe ataxia as early as 10-12 weeks after birth. Mechanistically, we find that Ronin is part of a protein complex in the cerebellum that is distinct from the one previously found in embryonic stem cells. Importantly, ectopically expressed Ronin raises the protein level of Ataxin-1 (Atxn1), the causative gene of the most common type of SCA, SCA1. Hence, our data provide evidence for a link between Ronin and SCAs, and also suggest that Ronin may be involved in the development of other neurodegenerative diseases.

Spinocerebellar ataxias (SCAs) are a group of genetically heterogeneous inherited neurodegenerative disorders characterized by progressive ataxia and cerebellar degeneration. Here, we used a mouse model to test a possible connection between SCA and Ronin (Thap11), a polyglutamine-containing transcriptional regulator encoded in a region of human chromosome 16q22.1 that has been genetically linked to SCA type 4. We report that transgenic expression of Ronin in mouse cerebellar Purkinje cells leads to detrimental loss of these cells and the development of severe ataxia as early as 10 weeks after birth. Mechanistically, we find that several SCA-causing genes harbor Ronin DNA-binding motifs and are transcriptionally deregulated in transgenic animals. In addition, ectopic expression of Ronin in embryonic stem cells significantly increases the protein level of Ataxin-1, the protein encoded by Atxn1, alterations of which cause SCA type 1. This increase is also seen in the cerebellum of transgenic animals, although the latter was not statistically significant. Hence, our data provide evidence for a link between Ronin and SCAs, and suggest that Ronin may be involved in the development of other neurodegenerative diseases., Summary: Ronin is a polyglutamine protein encoded in a region of human chromosome 16q22.1 linked to spinocerebellar ataxia type 4. Overexpression of Ronin in mouse cerebellar Purkinje cells leads to their loss and ataxia.

Summary Ronin (THAP11), a DNA-binding protein that evolved from a primordial DNA transposon by molecular domestication, recognizes a hyperconserved promoter sequence to control developmentally and metabolically essential genes in pluripotent stem cells. However, it remains unclear whether Ronin or related THAP proteins perform similar functions in development. Here, we present evidence that Ronin functions within the nascent heart as it arises from the mesoderm and forms a four-chambered organ. We show that Ronin is vital for cardiogenesis during midgestation by controlling a set of critical genes. The activity of Ronin coincided with the recruitment of its cofactor, Hcf-1, and the elevation of H3K4me3 levels at specific target genes, suggesting the involvement of an epigenetic mechanism. On the strength of these findings, we propose that Ronin activity during cardiogenesis offers a template to understand how important gene programs are sustained across different cell types within a developing organ such as the heart., Graphical Abstract, Highlights • Ronin displays complex expression patterns during embryogenesis • Ronin is critical for heart growth • Ronin regulates genetic growth programs • Ronin binding influences H3K4me3 levels at target genes, Fujita et al. find that the transcriptional regulator Ronin (Thap11) appears to control embryonic heart development. Early heart-specific knockout of Ronin leads to growth defects in the developing heart and embryonic lethality, whereas slightly later Ronin loss results in severe dilated cardiomyopathy in the adult.

Summary THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression., Highlights • Wild-type THAP1 regulates ESC potential, survival, and proliferation • THAP1 is essential for ESC differentiation, particularly neuroectodermal • Thap1C54Y or ΔExon2 ESCs prolong expression of pluripotent genes upon differentiation • Thap1C54Y or ΔExon2 EBs show increased cell death and abnormal differentiation, Ehrlich and colleagues identified THAP1, mutations of which cause dystonia (DYT6), as an essential regulator of ESC survival, proliferation, and differentiation. As THAP1 mutations result in a neurologic disease, this work examined the effects of a causative mutation, C54Y, and an essentially null allele, ΔExon2, on neuronal differentiation.

SummaryA fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression.

Organoids-or pluripotent stem cell-derived in vitro-grown simplified mini organs-have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses. The protocol starts from stem cell-derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single-cycle lentivirus expressing a fluorescent reporter protein. © 2018 by John Wiley & Sons, Inc.

Scientific research on human neural stem cells and cerebral organoids has confirmed the congenital neurotropic and neurodestructive nature of the Zika virus. However, the extent to which prenatal ZIKV infection is associated with more subtle brain alterations, such as epigenetic changes, remains ill defined. Here, we address the question of whether ZIKV infection induces DNA methylation changes with the potential to cause brain disorders later in life., Zika virus (ZIKV) infection during early pregnancy can cause microcephaly and associated defects at birth, but whether it can induce neurologic sequelae that appear later in life remains unclear. Using a model of the developing brain based on embryonic stem cell-derived brain organoids, we studied the impact of ZIKV infection on the DNA methylation pattern across the entire genome in selected neural cell types. The virus unexpectedly alters the DNA methylome of neural progenitors, astrocytes, and differentiated neurons at genes that have been implicated in the pathogenesis of a number of brain disorders, most prominently mental retardation and schizophrenia. Our results suggest that ZIKV infection during fetal development could lead to a spectrum of delayed-onset neuropsychiatric complications. IMPORTANCE Scientific research on human neural stem cells and cerebral organoids has confirmed the congenital neurotropic and neurodestructive nature of the Zika virus. However, the extent to which prenatal ZIKV infection is associated with more subtle brain alterations, such as epigenetic changes, remains ill defined. Here, we address the question of whether ZIKV infection induces DNA methylation changes with the potential to cause brain disorders later in life.

Dystonia is characterized by involuntary muscle contractions. Its many forms are genetically, phenotypically and etiologically diverse and it is unknown whether their pathogenesis converges on shared pathways. Mutations in THAP1 [THAP (Thanatos-associated protein) domain containing, apoptosis associated protein 1], a ubiquitously expressed transcription factor with DNA binding and protein-interaction domains, cause dystonia, DYT6. There is a unique, neuronal 50-kDa Thap1-like immunoreactive species, and Thap1 levels are auto-regulated on the mRNA level. However, THAP1 downstream targets in neurons, and the mechanism via which it causes dystonia are largely unknown. We used RNA-Seq to assay the in vivo effect of a heterozygote Thap1 C54Y or ΔExon2 allele on the gene transcription signatures in neonatal mouse striatum and cerebellum. Enriched pathways and gene ontology terms include eIF2α Signaling, Mitochondrial Dysfunction, Neuron Projection Development, Axonal Guidance Signaling, and Synaptic LongTerm Depression, which are dysregulated in a genotype and tissue-dependent manner. Electrophysiological and neurite outgrowth assays were consistent with those enrichments, and the plasticity defects were partially corrected by salubrinal. Notably, several of these pathways were recently implicated in other forms of inherited dystonia, including DYT1. We conclude that dysfunction of these pathways may represent a point of convergence in the pathophysiology of several forms of inherited dystonia., Author summary Dystonia is a brain disorder that causes disabling involuntary muscle contractions and abnormal postures. Mutations in THAP1, a zinc-finger transcription factor, cause DYT6, but its neuronal targets and functions are unknown. In this study, we sought to determine the effects of Thap1C54Y and ΔExon2 alleles on the gene transcription signatures at postnatal day 1 (P1) in the mouse striatum and cerebellum in order to correlate function with specific genes or pathways. Our unbiased transcriptomics approach showed that Thap1 mutants revealed multiple signaling pathways involved in neuronal plasticity, axonal guidance, and oxidative stress response, which are also present in other forms of dystonia, particularly DYT1. We conclude that dysfunction of these pathways may represent a point of convergence on the pathogenesis of unrelated forms of inherited dystonia.

SummaryBasal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A). These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications.

Parkinson's disease is associated with the loss of dopaminergic neurons in the midbrain. Clinical studies investigating replacement of these neurons with in vitro-generated neurons are currently underway. However, this approach has been limited by difficulties in scaling up on-demand production of midbrain dopaminergic (mDA) neurons from pluripotent stem cells. Cryo-preservation may offer a solution, as it allows for banking of quality controlled mDA neurons. In this study, we tested different freezing conditions and found that optimal cryopreservation of immature human mDA neurons at an early differentiation time point was achieved in STEM-CELLBANKER medium using a controlled freezing program. Key words: Parkinson's disease, Cryopreservation, Midbrain dopaminergic neurons, Human induced pluripotent stem cells, 6-OHDA mouse model

Keeping Cells Cooperating Multicellular organisms have certain advantages over those that are single-celled. To evolve, however, they must surmount a persistent challenge: ensuring that their constituent cells cooperate with one another rather than “cheat” or compete with each other for resources. Dejosez et al. (p. 1511 , published online 12 September) performed a genome-wide screen in induced pluripotent stem cells to search for genes that promote cell cooperation. A number of genes were identified of which knockdown allowed competitive behavior to dominate. These genes formed a network centered on p53, topoisomerase 1, and olfactory receptors. Thus, a genetic mechanism may promote cooperation in the developing embryo.

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation. DOI: http://dx.doi.org/10.7554/eLife.12175.001, eLife digest Scientific investigation requires researchers to use experimental observations to understand the biological process that resulted in these observations. One example is a cellular process called transcription, where the DNA of a gene is copied many times to make molecules of messenger RNA (mRNA), which are later used as instructions to make proteins. Scientists indirectly measure the dynamics of transcription, that is, how often the gene produces mRNA, by counting how many mRNA molecules there are in many individual cells. These numbers are then compared to the predictions made by a mathematical model of transcription, and if the model and experiment agree well, this is interpreted to mean that the model properly describes how often this gene is transcribed. Unfortunately, this procedure is not straightforward because many factors complicate the relationship between the dynamics of transcription and the number of mRNAs that will be detected in each cell at any one point in time. For example, it is not possible to tell whether a detected mRNA has just been transcribed, or whether it was made hours ago. The age of the cell and how many copies of the template DNA are present also affect the dynamics of transcription. As a result, mRNA measurements may be misinterpreted, leading to wrong conclusions about how highly particular genes are transcribed. To address this problem, Skinner et al. first improved the experimental measurements by discriminating between mature mRNA and the new mRNA that is still being transcribed. The experiments also measured how much DNA each cell contains, which indicates how old the cell is. These measurements were incorporated into a new mathematical model that is able to predict the dynamics of transcription of particular genes. Skinner et al. applied their method to two mouse genes called Oct4 and Nanog, which regulate the transformation of embryonic stem cells into other types of cells. The experiments show that both genes can switch between an “on” state where they are being actively transcribed and an “off” state where little or no mRNA is being produced. However, Nanog switches between these two states less often than Oct4, which results in larger variations between the numbers of mRNAs between different cells. The experiments also show that over the life of the cell, the level of transcription from each copy of the DNA decreases. Skinner et al.’s approach can be used to refine our knowledge of the transcription of other genes. However, to further improve our understanding of transcription, measurements of other factors will need to be incorporated into the mathematical models. DOI: http://dx.doi.org/10.7554/eLife.12175.002

Summary Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state., Graphical Abstract Highlights ► Knockout of Blimp1 has no effect on derivation or maintenance of ESCs or epiSCs ► Blimp1 is not required for reversion of epiSCs to an ESC state ► Blimp1 is required for PGC specification ► Transit through a PGC state is not obligatory for acquisition of pluripotency, Bao et al. demonstrate that the key determinant of primordial germ cells (PGCs) Blimp1 is dispensable for the derivation of embryonic stem cells (ESCs) and epiblast stem cells (epiSCs) and for reprogramming. This suggests that acquisition of pluripotency does not entail an obligatory route through a Blimp1-positive PGC-like state.

Abundant cell death marks early embryonic development. New work reported in Developmental Cell from Diaz-Diaz and colleagues (2017) proposes that this death results from cell competition arising from differences in cellular differentiation status, thus providing a physiological mechanism for controlling the make-up of the pluripotent stem cell population.

The ability to become nearly any cell type is restricted to eggs, sperm and primitive stem cells in very early embryos. Two studies reveal that maintaining this pluripotent state in vitro comes at a cost. See Letters & MEK1/2 and GSK3β inhibitors enhance the derivation of mouse embryonic stem cells (ES cells) in a ground state of pluripotency that closely resembles the inner cell mass of the pre-implantation embryo. However, the effect of long-term culture under these conditions on the developmental potential of ES cells is unclear. Konrad Hochedlinger and colleagues show that these culture conditions lead to detrimental changes in epigenetic features, genomic stability and in vivo developmental potential. The authors find that MEK inhibitors trigger changes in DNA methyltransferase expression that are linked to the observed abnormalities and show that using a Src inhibitor instead prevents the development of these abnormalities, preserving the genomic integrity and developmental potential of the ES cells. Elsewhere in this issue, Yashuiro Yamada and colleagues show that derivation of female ES cells under these culture conditions induces a loss of DNA methylation and erasure of genomic imprints, which are not recovered following differentiation. The team also develops a culture medium that preserves genomic features of a female ES cell. MEK1/2 and GSK3β inhibitors enhance derivation of mouse embryonic stem cells (ES cells) in a ground state of pluripotency that closely resembles the inner cell mass of the pre-implantation embryo. However, the effect of long-term culture under these conditions on the developmental potential of ES cells is unclear. Yashuiro Yamada and colleagues show that derivation of female ES cells under these conditions induces a loss of DNA methylation and erasure of genomic imprints, which are not recovered following differentiation. In parallel, the authors show that 2i-ES cells have impaired embryonic and placental development. The group also develops a culture medium that preserves the genomic features of female 2i-ES cells. Elsewhere in this issue, Konrad Hochedlinger and colleagues also show how these culture conditions lead to detrimental changes in epigenetic features, genomic stability and in vivo developmental potential and suggest an alternative inhibitor that preserves the genomic integrity of the ES cells.

δ-Catenin is an Armadillo protein of the p120-catenin subfamily capable of modulating cadherin stability, small GTPase activity, and nuclear transcription. From yeast two-hybrid screening of a human embryonic stem cell cDNA library, we identified δ-catenin as a potential interacting partner of the caspase-3 protease, which plays essential roles in apoptotic as well as non-apoptotic processes. Interaction of δ-catenin with caspase-3 was confirmed using cleavage assays conducted in vitro, in Xenopus apoptotic extracts, and in cell line chemically induced contexts. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo repeat 6 of δ-catenin, was identified through peptide sequencing. Cleavage thus generates an amino-terminal (residues 1–816) and carboxyl-terminal (residues 817–1314) fragment, each containing about half of the central Armadillo domain. We found that cleavage of δ-catenin both abolishes its association with cadherins and impairs its ability to modulate small GTPases. Interestingly, 817–1314 possesses a conserved putative nuclear localization signal that may facilitate the nuclear targeting of δ-catenin in defined contexts. To probe for novel nuclear roles of δ-catenin, we performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating interaction with an uncharacterized KRAB family zinc finger protein, ZIFCAT. Our results indicate that ZIFCAT is nuclear and suggest that it may associate with DNA as a transcriptional repressor. We further determined that other p120 subfamily catenins are similarly cleaved by caspase-3 and likewise bind ZIFCAT. Our findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120-catenin subfamily members, facilitating the coordinate modulation of cadherins, small GTPases, and nuclear functions.

There is intense contemporary interest in the identity, stability, and potential of stem cells, in the body or in the lab. The unusually comprehensive view provided by the 7th annual meeting of the ISSCR provides a framework to summarize recent progress.

Pluripotency is a unique biological state that allows cells to differentiate into any tissue in the body. Here we describe a novel candidate pluripotency factor, Ronin, that acts independently of canonical transcription factors and possesses a THAP domain, which is associated with sequence-specific DNA binding and epigenetic silencing of gene expression. Ronin is expressed primarily during the earliest stages of murine embryonic development, and its deficiency in mice produces periimplantational lethality and defects in the inner cell mass. Ronin ablation by a conditional knockout strategy prevents the growth of ES cells. Most critically, forced expression of Ronin allows ES cells to proliferate without differentiation under conditions that normally do not promote self-renewal, and it partly compensates for the effects of Oct4 knockdown. We demonstrate that Ronin binds directly to the HCF-1 protein, a key regulator of transcriptional control. Our findings identify Ronin as an essential factor underlying embryogenesis and ES cell pluripotency. Its direct binding to HCF-1 supports an epigenetic mechanism of gene repression in pluripotent cells.

SummaryEmbryonic stem cells (ESCs) are capable of indefinite self-renewal while retaining the ability to differentiate to any of the three germ layers that give rise to all somatic cell types. An emerging view is that a core set of transcription factors, including Oct4, Sox2, and Nanog, form a robust autoregulatory circuit that maintains ESCs in a self-renewing state. To accommodate the capacity of such cells to undergo germ layer-specific differentiation, we predicted a posttranslational mechanism that could negatively regulate these core self-renewal factors. Here we report caspase-induced cleavage of Nanog in differentiating ESCs. Stem cells lacking the Casp3 gene showed marked defects in differentiation, while forced expression of a caspase cleavage-resistant Nanog mutant in ESCs strongly promoted self-renewal. These results link a major component of the programmed cell-death pathway to the regulation of ESC development.

Described here are recent discoveries in my laboratory which suggest that the complement of factors needed to direct ES cell pluripotency may be considerably larger than originally thought and may include proteins that act independently of the canonical factors described thus far. They also provide insight into a novel screening method that could be used to accelerate the identification and characterization of such factors.

Because embryonic stem (ES) cells are generally derived by the culture of inner cell mass (ICM) cells, they are often assumed to be the equivalent of ICM cells. However, various evidence indicates that ICM cells transition to a different cell type during ES-cell derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm cells derived directly from the ICM. However, differences between ES cells and primitive ectoderm cells have caused developmental biologists to question whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue culture environment. Here, we review recent evidence that the closest in vivo equivalent of an ES cell is an early germ cell.

The excitement and controversy surrounding the potential role of human embryonic stem (ES) cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-beta (TGF-beta) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.

Abstract —Infiltration of monocytes into the arterial wall is an early cellular event in atherogenesis. Recent evidence shows that C-reactive protein (CRP) is deposited in the arterial intima at sites of atherogenesis. In this study, we demonstrate that CRP deposition precedes the appearance of monocytes in early atherosclerotic lesions. CRP is chemotactic for freshly isolated human blood monocytes. A specific CRP receptor is demonstrated on monocytes in vitro as well as in vivo, and blockage of the receptor by use of a monoclonal anti-receptor antibody completely abolishes CRP-induced chemotaxis. CRP may play a major role in the recruitment of monocytes during atherogenesis.

Tiam1 activates the Rho-like GTPase Rac1, and studies indicate that Tiam1–Rac1 signaling affects invasion in different ways depending on the cell type studied. However, no investigations on Tiam1 in human tumors have been reported. Here, we show that for 4 of 5 human renal-cell carcinoma (RCC) cell lines the expression levels of Tiam1 tended to be inversely correlated with in vitro invasiveness, whereas no obvious correlation could be found between the expression levels of Rac1 and invasion. Subsequent mutation analysis of these cell lines revealed no mutations in Rac1 but up to 5 different point mutations in the Tiam1 gene. Of these, 1 mutation (A441G) was located in the NH2-terminal pleckstrin homology domain, which is essential for membrane localization and functional activity of Tiam1. By analysis of an additional 30 primary human RCCs, mutation A441G was found in 4 of 35 tumors and tumor cell lines (11.5%) but not in the respective normal kidney tissues. By enzymatic digestion, mutation A441G proved to be heterozygous, suggesting a dominant active function. This was supported by showing that stable over-expression of mutated A441G-Tiam1 induced transformation of NIH3T3 cells, as determined in a colony formation assay, whereas empty vector and wild-type Tiam1 failed to do so. In conclusion, a distinct Tiam1 mutation (A441G) was identified in several human RCCs. This mutation induced transformation of NIH3T3 cells and, hence, might play a major role in the progression of human RCCs. Further analyses on Tiam1 mutations in human tumors might give new clues to their role in tumor progression. Int. J. Cancer 88:369–376, 2000. © 2000 Wiley-Liss, Inc.

Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that germ cell nuclear factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly reprogrammed, allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not reprogrammed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells recapitulated the phenotype of Gcnf knockout (Gcnf−/−) ES cells. Reintroduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf−/− ES cells rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely reprogrammed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells. STEM Cells 2013;31:2659–2666

Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse; however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here, we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem cell (ESC) differentiation as a model of early development. During our study of GCNF knockout (GCNF−/−) ESC, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ESC differentiation. This was determined to be an indirect effect of deregulation Mir302a, which is a cyclin D1 suppressor via binding to the 3′UTR of cyclin D1 mRNA. Moreover, we showed that Mir302 is a target gene of GCNF that inhibits Mir302 expression by binding to a DR0 element within its promoter. Inhibition of Mir302a using Mir302 inhibitor during differentiation of GCNF−/− ESCs restored cyclin D1 expression. Similarly over-expression of GCNF during differentiation of GCNF−/− ESCs rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a. Stem Cells 2014;32:1527–1537

Publisher Summary This chapter discusses the three genes whose overall targeting frequencies suggest that homologous recombination is a broadly applicable technique in human ES cells. The homologous recombination frequencies are roughly comparable to those observed for mouse ES cells and suggest that, although successful transfection strategies differ between human and mouse ES cells, homologous recombination itself may be similar. Homologous recombination in human ES cells will be important for studying gene function in vitro, and for lineage selection. It is a powerful approach for understanding the function of any human gene. For therapeutic applications in transplantation medicine, controlled modification of specific genes should be useful for purifying specific ES cell-derived, differentiated cell types from a mixed population, and for altering the antigenicity of ES cell derivatives. It should be possible to give cells new properties (such as viral resistance) to combat specific diseases. Homologous recombination in human ES cells might also be used for recently described approaches combining therapeutic cloning with gene therapy. Another good example of where human ES cells and homologous recombination will be useful is in understanding the function of the human heart. With homologous recombination, one could generate human ES cell lines bearing mutations or polymorphisms in specific ion channels, and use ES cell-derived cardiomyocytes to better understand the effects of the mutations on the physiology of the heart. A panel of ion channel polymorphisms should be extremely useful for screening drugs for toxic side effects on the heart prior to clinical trials. Homologous recombination in human ES cells would be useful for numerous in vitro models of human disease.

Homologous recombination applied to mouse embryonic stem (ES) cells has revolutionized the study of gene function in mammals. Although most often used to generate knockout mice, homologous recombination has also been applied in mouse ES cells allowed to differentiate in vitro. Homologous recombination is an essential technique if human ES cells are to fulfill their promise as a basic research tool. It also has important implications for ES cell-based transplantation and gene therapies. Significant differences between mouse and human ES cells have hampered the development of homologous recombination in human ES cells. High, stable transfection efficiencies in human ES cells have been difficult to achieve, and, in particular, electroporation protocols established for mouse ES cells work poorly in human ES cells. Also, in contrast to their murine counterparts, human ES cells cannot be cloned efficiently from single cells, making it difficult to screen for rare recombination events. Here we report an electroporation approach, based on the physical characteristics of human ES cells, that we used to successfully target HPRT1, the gene encoding hypoxanthine phosphoribosyltransferase-1 (HPRT1), and POU5F1, the gene encoding octamer-binding transcription factor 4 (Oct4; also known as POU domain, class 5, transcription factor 1 (POU5F1)).

Pluripotency is a “blank” cellular state characteristic of specific cells within the early embryo (e.g., epiblast cells) and of certain cells propagated in vitro (e.g., embryonic stem cells, ESCs). The terms pluripotent cell and stem cell are often used interchangeably to describe cells capable of differentiating into multiple cell types. In this review, we discuss the prevailing molecular and functional definitions of pluripotency and the working parameters employed to describe this state, both in the context of cells residing within the early embryo and cells propagated in vitro.

Retroviral vectors remain the most efficient and widely applied system for induction of pluripotency. However, mutagenic effects have been documented in both laboratory and clinical gene therapy studies, principally as a result of dysregulated host gene expression in the proximity of defined integration sites. Here, we report that cells with characteristics of pluripotent stem cells can be produced from normal human fibroblasts in the absence of reprogramming transcription factors (TFs) during lentiviral (LV) vector-mediated gene transfer. This occurred via induced alterations in host gene and microRNA (miRNA) expression and detrimental changes in karyotype. These findings demonstrate that vector-induced genotoxicity may alone play a role in somatic cell reprogramming derivation and urges caution when using integrating vectors in this setting. Clearer understanding of this process may additionally reveal novel insights into reprogramming pathways.

A recent report1 suggests yet another application for embryonic stem (ES) cell technology: using such cells to immunize against cancer. However, before this novel strategy can prove useful, several issues need to be resolved. ES cells derived from preimplantation embryos2 and induced pluripotent stem (iPS) cells obtained by transient overexpression of specific transcription factors in somatic cells3,4,5 are frequently said to hold the potential to revolutionize biomedical research by providing new substrates for biological studies designed to understand human diseases, by offering cellular transplantation and replacement therapies, and by serving as a platform for pioneering drug development and screening efforts. However, as the history of biomedical research has made clear, the most triumphant breakthroughs have often emerged from areas initially not considered promising by most researchers.

Background: Smooth muscle cell (SMC) proliferation in atherosclerosis is regulated through the interaction of growth factors like platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor-1 (IGF-1) and their receptors (R). We hypothesized that serum starvation of SMCs may affect PDGFβ-R and IGF-1-R expression and, consequently, the effect of their cognate ligands on SMC survival/proliferation. Methods and Results: Serum starvation significantly increases PDGFβ-R but not IGF-1-R mRNA and protein expression in SMCs. PDGF-BB stimulates cell survival but not proliferation in serum-starved SMCs of the synthetic phenotype, whereas SMCs of the contractile phenotype respond to PDGF-BB by a significant increase in proliferation. Immunohistochemical analysis of coronary atherosclerotic lesions reveals PDGFβ-R expression in SMCs in the lamina fibromuscularis, but not in the media and in healthy parts of the arterial wall. No such differential expression was observed for IGF-1-R. Conclusions: Differential regulation of PDGFβ-R and IGF-1-R expression by serum starvation might represent a mechanism for the control of SMC survival/proliferation in atherogenesis and restenosis. The distribution of PDGFβ-Rs and IGF-1-Rs in atherosclerotic lesions may indicate an effect of serum starvation on SMCs in the arterial wall.

Embryonic (ES) stem cells can be expanded indefinitely, yet retain the ability to form all cell types of the body. Here we report that human ES cells differentiate exclusively by symmetric cell division in each of four distinct differentiation conditions examined. This suggests that, in some respects, ES cells more closely resemble precursor or transit amplifying cells rather than adult stem cells.

Two counteracting processes determine accumulation of human vascular smooth muscle cells (SMCs) in atherosclerotic lesions: cell proliferation and apoptosis. SMCs synthesize insulin-like growth factor-1 (IGF-1), which potently inhibits apoptosis. The terminal complement complex C5b-9 interacts with SMCs in early human atherogenesis. In this study, we investigated whether C5b-9 may activate the IGF-1 system in SMCs, resulting in the inhibition of SMC apoptosis. C5b-9 generation on SMCs in vitro markedly reduced CD95-mediated apoptosis as assessed by flowcytometric analysis of annexin V binding and in caspase 3 assays. C5b-9 induced both significant IGF-1 release and up-regulation of IGF-1 binding sites in SMCs. Immunoneutralization of IGF-1 with a monoclonal IGF-1 antibody abolished the antiapoptotic effects of C5b-9. We conclude that C5b-9 inhibits apoptosis in SMCs by inducing an autocrine IGF-1 loop. This mechanism may contribute to the accumulation of SMCs in early human atherosclerotic lesions.

The transcription factors Tcf3 and Nanog regulate many genes in embryonic stem cells, but according to two reports in this issue of Cell Stem Cell (Festuccia et al., 2012, Martello et al., 2012), only one, Esrrb , encoding an orphan nuclear hormone receptor, truly matters in the maintenance of self-renewal.

Background —LDL and C-reactive protein (CRP) are important cardiovascular risk factors. Both LDL and CRP deposit in the arterial wall during atherogenesis. Stranded LDL is taken up by macrophages, causing foam cell formation. Because native LDL does not induce foam cell formation, we hypothesized that CRP may opsonize native LDL for macrophages. Methods and Results —Monocytes were isolated from human blood and transformed into macrophages. CRP/LDL uptake was assessed by immunofluorescent labeling and the use of confocal laser scanning microscopy. Native LDL coincubated with CRP was taken up by macrophages by macropinocytosis. Uptake of the CRP/LDL coincubate was mediated by the CRP receptor CD32. Conclusions —We conclude that foam cell formation in human atherogenesis may be caused in part by uptake of CRP-opsonized native LDL.

One of the most fascinating and daunting challenges of contemporary developmental biology is to fully classify diverse cell types on the basis of a quantifiable genetic profile. Although compiling an inventory of such identifying features seems feasible for some specific cell types (e.g., those of the hematopoietic system), a robust and universal platform has yet to emerge. Indeed, in most cases, a series of confirmatory physiological assays are still needed to verify cell identity, yet such assays are available for only a small subset of classifiable cells and can be quite difficult to conduct and interpret. A study by Creyghton et al. (1) provides a possible solution to this conundrum. It gives us a first and unique glimpse of the regulatory complexity of enhancers at the epigenetic level and suggests that histone marks allow discrimination between at least two kinds of enhancers: those that are truly active and whose activities correlate closely and specifically with cell identity and those that are poised to engage in future enhancer activity, thus allowing predictions about the lineage decisions that the cell is facing.

Methods for generating embryonic-like stem cells have been established. The focus now is on finding ways to coax these cells into matching their natural counterparts as closely as possible, should this be desired.

Mouse lines with inducible reprogramming factors expressed from a single genomic locus will allow reprogramming studies in multiple cell types and defined genetic backgrounds.

A new report (Szutorisz et al., 2006) suggests that embryonic stem (ES) cells adopt an unusual strategy to remain perpetually poised for differentiation. Apparently, ES cells use the proteasome to target transcriptional preinitiation complexes, thus minimizing transcriptional noise at genes that promote differentiation.