Pluripotent Stem Cells

The differentiation of pluripotent stem cells as embryoid bodies (EBs) remains a common method for inducing differentiation toward many lineages. However, differentiation via EBs typically yields a significant amount of heterogeneity in the cell population, as most cells differentiate simultaneously toward different lineages, while others remain undifferentiated. Moreover, physical parameters, such as the size of EBs, can modulate the heterogeneity of differentiated phenotypes due to the establishment of nutrient and oxygen gradients. One of the challenges in examining the cellular composition of EBs is the lack of analytical methods that are capable of determining the phenotype of all of the individual cells that comprise a single EB. Therefore, the objective of this work was to examine the ability of a microfluidic cell trapping array to analyze the heterogeneity of cells comprising EBs during the course of early differentiation. The heterogeneity of single cell phenotype on the basis of protein expression of the pluripotent transcription factor OCT-4 was examined for populations of EBs and single EBs of different sizes at distinct stages of differentiation. Results from the cell trap device were compared with flow cytometry and whole mount immunostaining. Additionally, single cells from dissociated pooled EBs or individual EBs were examined separately to discern potential differences in the value or variance of expression between the different methods of analysis. Overall, the analytical method described represents a novel approach for evaluating how heterogeneity is manifested in EB cultures and may be used in the future to assess the kinetics and patterns of differentiation in addition to the loss of pluripotency.

Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks.

The induction of pluripotency in somatic cells is widely considered as a major breakthrough in regenerative medicine, because this approach provides the basis for individualized stem cell-based therapies. Moreover, with respect to cell transplantation and tissue engineering, expertise from bioengineering to transplantation medicine is now meeting basic research of stem cell biology.In this chapter, we discuss techniques, potential and possible risks of induced pluripotent stem (iPS) cells in the light of needs for patient-derived pluripotent stem cells. To this end, we compare these cells with other sources of pluripotent cells and discuss the first encouraging results of iPS cells in pharmacological research, disease modeling and cell transplantation, providing fascinating perspectives for future developments in biotechnology and regenerative medicine.

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REVIEW Cellular Therapy and Induced Neuronal Replacement for Huntington's Disease Abdellatif Benraiss & Steven A. Goldman ... 2011 Abstract Huntington's disease (HD) is an inherited, relentlessly progressive neurodegenerative disease with an invariably fatal outcome. ...

The differentiation potential of pluripotent embryonic stem cells (ESCs) can be manipulated via serum and medium conditions for direct cellular development or to maintain a naïve ground state. The selfrenewal state of ESCs can thus be induced by adding inhibitors of mitogen activated protein kinase (MAPK) and glycogen synthase kinase-3 (Gsk3), known as 2 inhibitors (2i) treatment. We have used a shotgun proteomics approach to investigate differences in protein expressions between 2i-and serum-grown mESCs. The results indicated that 164 proteins were significantly upregulated and 107 proteins downregulated in 2i-grown cells compared to serum. Protein pathways in 2i-grown cells with the highest enrichment were associated with glycolysis and gluconeogenesis. Protein pathways related to organ development were downregulated in 2i-grown cells. In serum-grown ESCs, protein pathways involved in integrin and focal adhesion, and signaling proteins involved in the actin cytoskeleton regulation were enriched. We observed a number of nuclear proteins which were mostly involved in selfrenewal maintenance and were expressed at higher levels in 2i compared to serum-Dnmt1, Map2k1, Parp1, Xpo4, Eif3g, Smarca4/Brg1 and Smarcc1/Baf155. Collectively, the results provided an insight into the key protein pathways used by ESCs in the ground state or metastable conditions through 2i or serum culture medium, respectively.

New research suggests that common pathways are altered in many neurodevelopmental disorders including autism spectrum disorder; however, little is known about early molecular events that contribute to the pathology of these diseases. The study of monogenic, neurodevelopmental disorders with a high incidence of autistic behaviours, such as fragile X syndrome, has the potential to identify genes and pathways that are dysregulated in autism spectrum disorder as well as fragile X syndrome. In vitro generation of human disease-relevant cell types provides the ability to investigate aspects of disease that are impossible to study in patients or animal models. Differentiation of human pluripotent stem cells recapitulates development of the neocortex, an area affected in both fragile X syndrome and autism spectrum disorder. We have generated induced human pluripotent stem cells from several individuals clinically diagnosed with fragile X syndrome and autism spectrum disorder. When different...

Significance DNA transposons that translocate by excision from a donor site and insertion into a target site are often used for genome engineering by insertional mutagenesis and transgenesis. The piggyBac element is especially useful because it can excise precisely from an insertion site, restoring the site to its pretransposon state. Precise excision is particularly useful when transient transgenesis is needed, for example, in the transient introduction of transcription factors for induced pluripotent stem cell production. We have used mutagenesis to generate an Excision + Integration − transposase that allows piggyBac excision without potentially harmful reintegration. These mutations likely lie in a target DNA-binding domain.

Understanding the fundamental mechanisms of tumorigenesis remains one of the most pressing problems in modern biology. To this end, stem-like cells with tumor-initiating potential have become a central focus in cancer research. While the cancer stem cell hypothesis presents a compelling model of self-renewal and partial differentiation, the relationship between tumor cells and normal stem cells remains unclear. We identify, in an unbiased fashion, mRNA transcription patterns associated with pluripotent stem cells. Using this profile, we derive a quantitative measure of stem cell-like gene expression activity. We show how this 189 gene signature stratifies a variety of stem cell, malignant and normal tissue samples by their relative plasticity and state of differentiation within Concordia, a diverse gene expression database consisting of 3,209 Affymetrix HGU133+ 2.0 microarray assays. Further, the orthologous murine signature correctly orders a time course of differentiating embryoni...

The function of the proteasome is essential for maintenance of cellular homeostasis, and in pluripotent stem cells, this has been extended to the removal of nascent proteins in a manner that restricts differentiation. In this study, we show enhanced expression of genes encoding subunits of the 20S proteasome in human embryonic stem cells (hESCs) coupled to their downregulation as the cells progress into differentiation. The decrease in expression is particularly marked for the alternative catalytic subunits of the 20S proteasome variant known as the immunoproteasome indicating the possibility of a hitherto unknown function for this proteasome variant in pluripotent cells. The immunoproteasome is normally associated with antigen-presenting cells where it provides peptides of an appropriate length for antibody generation; however, our data suggest that it may be involved in maintaining the pluripotency in hESCs. Selective inhibition of two immunoproteasome subunits (PSMB9 and PSMB8) results in downregulation of cell surface and transcriptional markers that characterize the pluripotent state, subtle cell accumulation in G1 at the expense of S-phase, and upregulation of various markers characterizing the differentiated primitive and definitive lineages arising from hESC. Our data also support a different function for each of these two subunits in hESC that may be linked to their selectivity in driving proteasome-mediated degradation of cell cycle regulatory components and/or differentiation inducing factors.

Background: Inactivation of one X chromosome is established early in female mammalian development and can be reversed in vivo and in vitro when pluripotency factors are re-expressed. The extent of reactivation along the inactive X chromosome (Xi) and the determinants of locus susceptibility are, however, poorly understood. Here we use cell fusion-mediated pluripotent reprograming to study human Xi reactivation and allele-specific single nucleotide polymorphisms (SNPs) to identify reactivated loci. Results: We show that a subset of human Xi genes is rapidly reactivated upon re-expression of the pluripotency network. These genes lie within the most evolutionary recent segments of the human X chromosome that are depleted of LINE1 and enriched for SINE elements, predicted to impair XIST spreading. Interestingly, this cadre of genes displays stochastic Xi expression in human fibroblasts ahead of reprograming. This stochastic variability is evident between clones, by RNA-sequencing, and at the single-cell level, by RNA-FISH, and is not attributable to differences in repressive histone H3K9me3 or H3K27me3 levels. Treatment with the DNA demethylating agent 5-deoxy-azacytidine does not increase Xi expression ahead of reprograming, but instead reveals a second cadre of genes that only become susceptible to reactivation upon induction of pluripotency. Conclusions: Collectively, these data not only underscore the multiple pathways that contribute to maintaining silencing along the human Xi chromosome but also suggest that transcriptional stochasticity among human cells could be useful for predicting and engineering epigenetic strategies to achieve locus-specific or domain-specific human Xi gene reactivation.

No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is, however, limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel), 0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker, MATH5. Furthermore, 0.5% RGD-alginate hydrogel encapsulation did n...

Background: The surge in the number of gene expression studies and tendencies to increase the quality of analysis have necessitated the identification of stable reference genes. Although rabbits are classical experimental model animals, stable reference genes have not been identified for normalization. The aims of this study were to compare the expression profiles of the widely used reference genes in rabbit oocytes and preimplantation stage embryos, and to select and validate stable ones to use as reference. Results: Quantitative real time PCR method was used to evaluate 13 commonly used references (Actb,

Accurately predicting cardioactive effects of new molecular entities for therapeutics remains a daunting challenge. Immense research effort has been focused toward creating new screening platforms that utilize human pluripotent stem cell (hPSC)-derived cardiomyocytes and three-dimensional engineered cardiac tissue constructs to better recapitulate human heart function and drug responses. As these new platforms become increasingly sophisticated and high throughput, the drug screens result in larger multidimensional datasets. Improved automated analysis methods must therefore be developed in parallel to fully comprehend the cellular response across a multidimensional parameter space. Here, we describe the use of machine learning to comprehensively analyze 17 functional parameters derived from force readouts of hPSC-derived ventricular cardiac tissue strips (hvCTS) electrically paced at a range of frequencies and exposed to a library of compounds. A generated metric is effective for th...

Cornelia de Lange syndrome (CdLS) is a complex disorder with multiple structural and developmental defects caused by mutations in structural and regulatory proteins involved in the cohesin complex. NIPBL, a cohesin regulatory protein, has been identified as a critical protein responsible for the orchestration of transcriptomic regulatory networks necessary for embryonic development. Mutations in NIPBL are responsible for the majority of cases of CdLS. Through RNA-sequencing of human induced pluripotent stem cells and in vitro-derived cardiomyocytes, we identified hundreds of mRNAs, pseudogenes, and non-coding RNAs with altered expression in NIPBL+/- patient-derived cells. We demonstrate that NIPBL haploinsufficiency leads to upregulation of gene sets identified in functions related to nucleosome, chromatin assembly, RNA modification and downregulation of Wnt signaling, cholesterol biosynthesis and vesicular transport in iPSC and cardiomyocytes. Mutations in NIPBL result in the dysre...

KEY REFERENCE An excellent history of gene targeting, the history of the CRISPR/Cas9 system and the future of CRISPR/Cas9 mediated genome engineering (Doudna and Charpentier, 2014). Several protocol papers have been published over the last several years providing detailed instructions for carrying out gene targeting experiments (Dambournet et al., 2014; Ran et al., 2013b; Song et al., 2014). For a review on TALEN and CRISPR/Cas9 mediated gene targeting and the advantages and disadvantages of each method, we refer readers to a recent review article (Hendriks et al., 2016). Several other reviews provide a basic overview of the CRISPR/Cas9 system and its future applications (Hsu et al., 2014; Zhang et al., 2014a). INTERNET RESOURCES CRISPR Design Tool (Feng Zhang Laboratory) http://crispr.mit.edu/ This tool is useful for the design of CRISPR sgRNAs. It allows for the design of both nucleases and paired nickases. For detailed instructions of use, refer to the help tab. CHOPCHOP CRISPR Design Tool (Alex Schier laboratory) https://chopchop.rc.fas.harvard.edu/ Resource for the design of sgRNAs that provides a helpful visual interface. It also provides PCR primers that can be used for the SURVEYOR assay, Sanger sequencing of the targeted region or the in vitro cutting assay with Cas9 protein for a wide range of organisms.

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infections. Inappropriate migration also underpins the formation of metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate that we can use this system to study the molecular control of stem cell migration and show that snail-1, snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions without local stem cells, in the absence of injury, maintaining tissue h...

The environmental cues received by the cells from synthetic substrates in vitro are very different from those they receive in vivo. In this study, we applied the Langmuir-Schaefer (LS) deposition, a variant of Langmuir-Blodgett technique, to fabricate a biomimetic microenvironment mimicking the structure and organization of native Bruch's membrane for the production of the functional human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cells. Surface pressure-area isotherms were measured simultaneously with Brewster angle microscopy to investigate the self-assembly of human collagens type I and IV on air-subphase interface. Furthermore, the structure of the prepared collagen LS films was characterized with scanning electron microscopy, atomic force microscopy, surface plasmon resonance measurements and immunofluorescent staining. The integrity of hESC-RPE on double layer LS films was investigated by measuring transepithelial resistance and permeability of smal...

Many signals must be integrated to maintain selfrenewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylationmediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming. RESULTS Loss-of-Function shRNA Screen Identifies Protein Kinases and Phosphatases Required for ESC Self-Renewal and Pluripotency PKase-and PPase-mediated phosphorylation and dephosphorylation events control a wide range of biological processes. To

Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties whi...

Oct4 and Sox2 transcription factors (belonging to the Yamanaka's factor family) and Nanog, named together as core transcription factors of pluripotency, are indispensable to induce and maintain the pluripotency state. They act generally as activators of genes coding for transcription factors, cofactors and chromatin regulators. They also activate microRNA expression. In addition, Oct4, Sox2 and Nanog function as repressors of genes for factors responsible for escape from pluripotency and differentiation. Core transcription factors positively regulate their own promoters, forming a positive-feedback loop. In recent times, researchers' attention has been attracted towards Oct4, Sox2 and Nanog as potential markers for cancer stem cells (CSCs). The expression of these factors has been confirmed in numerous types of tumors. The aim of this paper is to concisely review features of core transcription factors and their role in embryogenesis and tumorigenesis including the CSC hypoth...

Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium con...

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors, octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and v-myc myelocytomatosis viral oncogene homolog (C-MYC), can induce pluripotency in mouse and human fibroblasts. Here, we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A], C-MYC, and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology, in the ability to differentiate into cells of the 3 embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This, in turn, will allow us to better control cell-fate and apply this knowledge to cell therapy.

Human pluripotent stem cells (hPSCs) are attractive sources for regenerative medicine and disease modeling in vitro. Directed hPSC differentiation approaches have derived from knowledge of cell development in vivo rather than from stochastic cell differentiation. Moreover, there has been great success in the generation of 3D organ-buds termed 'organoids' from hPSCs; these consist of a variety of cell types in vitro that mimic organs in vivo. The organoid bears great potential in the study of human diseases in vitro, especially when combined with CRISPR/Cas9-based genome-editing. We summarize the current literature describing organoid studies with a special focus on kidney organoids, and discuss goals and future opportunities for organoid-based studies.

Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. ...

Clinical investigations using stem cell products in regenerative medicine are addressing a wide spectrum of conditions using a variety of stem cell types. To date, there have been few reports of safety issues arising from autologous or allogeneic transplants. Many cells administered show transient presence for a few days with trophic influences on immune or inflammatory responses. Limbal stem cells have been registered as a product for eye burns in Europe and mesenchymal stem cells have been approved for pediatric graft versus host disease in Canada and New Zealand. Many other applications are progressing in trials, some with early benefits to patients.

The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult. Pluripotent stem cell lines from the inner cell mass (ICM) 3 of the embryo, the so-called embryonic stem cells (ESC), were first established over 30 years ago from day 3.5 mouse (m) blastocysts (1, 2) and more recently from totipotent blastomeres (3, 4). These authentic mESC lines are dependent on LIF/STAT3 signaling for maintenance of pluripotency (5), tolerate complete enzymatic dispersal, and fulfill the stringent criteria of

To gain insight into mechanisms controlling SRY (sex determining region Y)-box 2 (Sox2) protein activity in mouse embryonic stem cells (ESCs), the endogenous Sox2 gene was tagged with FLAG/ Hemagglutinin (HA) sequences by homologous recombination. Sox2 protein complexes were purified from Sox2/FLAG/HA knockin ESCs, and interacting proteins were defined by mass spectrometry. One protein in the complex was poly ADP-ribose polymerase I (Parp1). The results presented below demonstrate that Parp1 regulates Sox2 protein activity. In response to fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling, Parp1 auto-poly ADP-ribosylation enhances Sox2-Parp1 interactions, and this complex inhibits Sox2 binding to octamer-binding transcription factor 4 (Oct4)/Sox2 enhancers. Based on these results, we propose a unique mechanism in which FGF signaling fine-tunes Sox2 activity through posttranslational modification of a critical interacting protein, Parp1, and balances the maintenance of ESC pluripotency and differentiation. In addition, we demonstrate that regulation of Sox2 activity by Parp1 is critical for efficient generation of induced pluripotent stem cells. regulatory pathway | gene targeting | tagged mice M ouse embryonic stem cells (ESCs), which are derived from

Cell based regenerative therapy has emerged as one of the most promising options of treatment for patients suffering from heart failure. Various adult stem cells types have undergone extensive clinical trials with limited success which is believed to be more of a cytokine effect rather than cell therapy. Pluripotent human embryonic stem cells (hESCs) have emerged as an attractive candidate stem cell source for obtaining cardiomyocytes (CMs) because of their tremendous capacity for expansion and unquestioned potential to differentiate into CMs. Studies carried out in animal models indicate that ES-derived CMs can partially remuscularize infarcted hearts and improve contractile function; however, the effect was not sustained over long follow up periods due to their limited capacity of cell division in vivo. Thus, the concept of transplanting multipotent cardiovascular progenitors derived from ES cells has emerged since the progenitors retain robust proliferative ability and multipoten...

Despite their abundance, the molecular functions of long non-coding RNAs in mammalian nervous systems remain poorly understood. Here we show that the long non-coding RNA, NEAT1, directly modulates neuronal excitability and is associated with pathological seizure states. Specifically, NEAT1 is dynamically regulated by neuronal activity in vitro and in vivo, binds epilepsy-associated potassium channel-interacting proteins including KCNAB2 and KCNIP1, and induces a neuronal hyper-potentiation phenotype in iPSC-derived human cortical neurons following antisense oligonucleotide knockdown. Next generation sequencing reveals a strong association of NEAT1 with increased ion channel gene expression upon activation of iPSC-derived neurons following NEAT1 knockdown. Furthermore, we show that while NEAT1 is acutely down-regulated in response to neuronal activity, repeated stimulation results in NEAT1 becoming chronically unresponsive in independent in vivo rat model systems relevant to temporal...

Endogenous naturally-occurring totipotent stem cells and pluripotent stem cells have been isolated from the skeletal muscle of adult mammals, including humans. This study was undertaken to determine their particular location within adult rat skeletal muscle and to verify the identity of the stained cells. Adult rats were euthanized following the guidelines of Mercer University's IACUC. Skeletal muscle was harvested and processed for immunocytochemistry. Cells were stained with carcinoembryonic antigen-cell adhesion molecule-1 (CEA-CAM-1) and stage-specific embryonic antigen-4 (SSEA-4). Positive and negative staining controls were run to verify the validity of the immunostaining. CEA-CAM-1+ cells were located preferentially within vascular connective tissues, whereas SSEA-4+ cells were located preferentially within neuronal connective tissues. CEA-CAM-1+ stem cells and SSEA-4+ cells were isolated from adult rat skeletal muscle, segregated, cloned from single cells, and their unique morphologies, differentiation potentials, and attributes in culture were subsequently characterized. Four populations of stem cells were identified that share the CEA-CAM-1 and/or SSEA-4 epitopes. Totipotent stem cells and transitional-totipotent stem cell/pluripotent stem cells share the CEA-CAM-1 epitope, whereas the transitional-totipotent stem cell/pluripotent stem cells, pluripotent stem cells, and transitional-pluripotent stem cell/germ layer lineage stem cells share the SSEA-4 epitope. This report describes native populations of endogenous naturally-occurring totipotent stem cells and pluripotent stem cells in adult rat skeletal muscle. Studies are ongoing to address their functional significance during normal tissue maintenance and repair of body organs.

Flatworms of the species are immortal-adult animals contain a large pool of pluripotent stem cells that continuously differentiate to all adult cell types. Therefore, single-cell transcriptome profiling of adult animals should reveal mature and progenitor cells. By combining perturbation experiments, gene expression analysis, a computational method that predicts future cell states from the transcriptional changes, and a lineage reconstruction method, we placed all major cell types onto a single lineage tree that connects all cells to a single stem cell compartment. We characterize gene expression changes during differentiation and discover cell types important for regeneration. Our results demonstrate the importance of single-cell transcriptome analysis for mapping and reconstructing fundamental processes of developmental and regenerative biology at high resolution.

of compounds showed distinct concentration-response profiles. Plotting of the lowest observed adverse effect concentrations (LOAEL) of the neuronal promoter activity against the general promoter activity or against cytotoxicity, allowed the differentiation between neurotoxic/ DNT substances and non-neurotoxic controls. Reporter activity responses in neurons were more susceptible to neurotoxic compounds than the reporter activities in ESCs from which they were derived. To relate the effective/toxic concentrations found in our study to relevant in vivo concentrations, we used a reverse pharmacokinetic modeling approach for three exemplary compounds (teriflunomide, geldanamycin, abiraterone). The dual luminescence reporter assay described in this study allows high-throughput, and should be particularly useful for the prioritization of the neurotoxic potential of a large number of compounds.

Both embryonic and adult stem cells are endowed with a superior capacity to prevent the accumulation of genetic lesions, repair them, or avoid their propagation to daughter cells, which would be particularly detrimental to the whole organism. Inducible pluripotent stem cells also display a robust DNA damage response, but the stability of their genome is often conditioned by the mutational history of the cell population of origin, which constitutes an obstacle to clinical applications. Cancer stem cells are particularly tolerant to DNA damage and fail to undergo senescence or regulated cell death upon accumulation of genetic lesions. Such a resistance contributes to the genetic drift of evolving tumors as well as to their limited sensitivity to chemo- and radiotherapy. Here, we discuss the pathophysiological and therapeutic implications of the molecular pathways through which stem cells cope with DNA damage.

Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement....

Highlights d Transcriptome profiling establishes a reference map for early mouse development d Lineage-specific RNA-seq captures the full dynamic range of gene expression d ESCs and diapaused and preimplantation epiblasts share common pluripotency circuitry

Specific cells within the early mammalian embryo have the capacity to generate all somatic lineages plus the germline. This property of pluripotency is confined to the epiblast, a transient tissue that persists for only a few days. In vitro, however, pluripotency can be maintained indefinitely through derivation of stem cell lines. Pluripotent stem cells established from the newly formed epiblast are known as embryonic stem cells (ESCs), whereas those generated from later stages are called postimplantation epiblast stem cells (EpiSCs). These different classes of pluripotent stem cell have distinct culture requirements and gene expression programs, likely reflecting the dynamic development of the epiblast in the embryo. In this chapter we review current understanding of how the epiblast forms and relate this to the properties of derivative stem cells. We discuss whether ESCs and EpiSCs are true counterparts of different phases of epiblast development or are culture-generated phenomen...

The propagation and maintenance of gene expression programs are at the foundation of the preservation of cell identity. A large and complex set of epigenetic mechanisms enables the long-term stability and inheritance of transcription states. A key property of authentic epigenetic regulation is being independent from the instructive signals used for its establishment. This makes epigenetic regulation, particularly epigenetic silencing, extremely robust and powerful to lock regulatory states and stabilise cell identity. In line with this, the establishment of epigenetic silencing during development restricts cell potency and maintains the cell fate choices made by transcription factors (TFs). However, how more immature cells that have not yet established their definitive fate maintain their transitory identity without compromising their responsiveness to signalling cues remains unclear. A paradigmatic example is provided by pluripotent embryonic stem (ES) cells derived from a transient population of cells of the blastocyst. Here, we argue that ES cells represent an interesting "epigenetic paradox": even though they are captured in a self-renewing state characterised by extremely efficient maintenance of their identity, which is a typical manifestation of robust epigenetic regulation, they seem not to heavily rely on classical epigenetic mechanisms. Indeed, self-renewal strictly depends on the TFs that previously instructed their undifferentiated identity and relies on a particular signalling-dependent chromatin state where repressive chromatin marks play minor roles. Although this "epigenetic paradox" may underlie their exquisite responsiveness to developmental cues, it suggests that alternative mechanisms to faithfully propagate gene regulatory states might be prevalent in ES cells.

Yolk sac tumours revisited. A review of their many faces and names We review the current knowledge on human yolk sac tumours (YSTs) 50 years after their initial description. Their complex nomenclature and histogenesis stress the fact that they are not a discrete entity, but represent a multifaceted group of neoplasms, for which the term primitive endodermal tumours would be more appropriate, accounting for their capacity to differentiate into various extraembryonal and somatic cell types. Different histological patterns of human YSTs correlate with the developmental potential of primitive endoderm and mesenchyme, but they are also similar to some murine experimental tumours. Exceptionally, YSTs replicate the tubular structures of the human yolk sac and allantois. Endodermal somatic differentiation reproduces pulmonary, intestinal and hepatic tissues and are identical with some, embryonal-type endodermal, gastric and lung carcinomas, which are indistinguishable from YSTs. YSTs may show an overgrowth of their mesenchymal (sarcomatous) and epithelial components (such as mucinous carcinoma or carcinoid) and also be a source of haematological malignancies. YSTs associated with non-germ cell tumours probably originate from malignant pluripotent somatic stem cells. Only AFP and glypican-3 are characteristic immunohistochemical markers. Pluripotent antibodies (SALL4, Lin28, IMP-3) help in differential diagnoses, while some differentiation markers (CDX2, TTF-1, HepPar1) facilitate recognition of unusual variants of YSTs.

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A) + transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, statespecific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.