Embryonic Stem Cell Populations Research Articles

Haspin-dependent and independent effects of the kinase inhibitor 5-Iodotubercidin on self-renewal and differentiation

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting “off-target” effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the “off-target” effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.

Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers.

The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine.

Evolution of the new head by gradual acquisition of neural crest regulatory circuits.

Summary:The neural crest is a vertebrate innovation proposed to be a key component of the “New Head” that imbued vertebrates with predatory behavior. To address how evolution of this cell type impacted the vertebrate body plan, we examined the molecular circuits that control neural crest development along the anteroposterior axis of a jawless vertebrate, the sea lamprey. Gene expression analysis showed that the lamprey cranial neural crest lacks most components of an amniote cranial-specific transcriptional circuit that confers the ability to form craniofacial cartilage onto other neural crest populations1. Consistent with this, hierarchical clustering revealed that the transcriptional profile of the lamprey cranial crest is more similar to the amniote trunk crest. Intriguingly, analysis of the cranial neural crest in little skate and zebrafish embryos demonstrated that the cranial-specific transcriptional circuit emerged via gradual addition of network components to the neural crest of gnathostomes, which subsequently became restricted to the cephalic region. Our results indicate that the ancestral neural crest at the base of vertebrates possessed a trunk-like identity. We propose that the emergence of the cranial neural crest, by progressive assembly of a novel axial-specific regulatory circuit, allowed for the elaboration of the New Head during vertebrate evolution.

Differential effects of fluoxetine and venlafaxine in the neural embryonic stem cell test (ESTn) revealed by a cell lineage map

There is a need for in vitro tests for the evaluation of chemicals and pharmaceuticals that may cause developmental neurotoxicity (DNT) in humans. The neural embryonic stem cell test (ESTn) is such an in vitro test that mimics early neural differentiation. The aim of this study was to define the biological domain of ESTn based on the expression of selective markers for certain cell types, and to investigate the effects of two antidepressants, fluoxetine (FLX) and venlafaxine (VNX), on neural differentiation. A cell lineage map was made to track neural differentiation and the effects of FLX and VNX in ESTn. Whole transcriptome analysis revealed differentiation from an embryonic stem cell population to a mixed culture of neural progenitors, neurons and neural crest cells 7 days into differentiation. Maturing neurons, astrocytes and oligodendrocytes were present after 13 days. Exposure to FLX or VNX led to different expression patterns between compounds at both time points. On day 7, both compounds upregulated most of the stem cell- and immature neuron markers, but had distinct effects on neural subtype markers. FLX downregulated glycinergic markers and upregulated cholinergic markers, while VNX had the opposite effect. On day 13, FLX and VNX affected their specific therapeutic targets, represented by mainly serotonergic markers by FLX- and dopaminergic and noradrenergic markers in VNX-exposed cultures, as well as oligodendrocyte and glycinergic neuron markers. This proof of concept study shows the added value of assessing DNT in ESTn through a cell lineage map and gives mechanistic insight in the potential neurodevelopmental effects of FLX and VNX. More compounds should be tested to further evaluate the use of the cell lineage map.

AMPK activators contribute to maintain naïve pluripotency in mouse embryonic stem cells

Pluripotent stem cells retain the property to self-renew and differentiate into all cell types under defined conditions. Among mouse embryonic stem cells (ESCs), which are pluripotent but heterogenous in gene expression and morphology, an ESC population cultured in small molecule inhibitors of two kinases, MAPK/ERK kinase (Mek) and Glycogen synthase kinase 3 (Gsk3), and leukemia inhibitory factor (Lif) (2i/L) is considered to be naïve pluripotent with uniform pluripotent machinery operation. Though the gene regulatory mechanism for the naïve pluripotency has been investigated in recent years, it is still not fully elucidated. Here we show a novel signaling involved in the maintenance of naïve pluripotency. An AMP-activated protein kinase (AMPK) activator, AICAR (5-Aminoimidazole-4-carboxamied-1-β-riboside) blocked the differentiation of mouse naïve ESCs in the absence of 2i/L and maintained the naïve state. AICAR with Lif condition induced an almost comparable level of naïve pluripotent gene expression in mouse ESCs. Another AMPK activator, A769662, also showed similar effects. A p38 inhibitor, SB203580, blocked the AMPK activation-elicited naïve state maintenance. On the other hand, p38 activation partially mimicked the maintenance effects of AMPK activators, suggesting that p38 is one of the functional downstream molecules to conduct the AMPK effects. Thus, AMPK pathway should be involved in the molecular circuitry of naïve pluripotency in mouse ESCs. These findings would be a valuable clue to further elucidate the molecular machinery of naïve pluripotency.

Bioflavonoids promote stable translocations between MLL-AF9 breakpoint cluster regions independent of normal chromosomal context: Model system to screen environmental risks.

Infant acute leukemias are aggressive and characterized by rapid onset after birth. The majority harbor translocations involving the MLL gene with AF9 as one of its most common fusion partners. MLL and AF9 loci contain breakpoint cluster regions (bcrs) with sequences hypothesized to be targets of topoisomerase II inhibitors that promote translocation formation. Overlap of MLL bcr sequences associated with both infant acute leukemia and therapy-related leukemia following exposure to the topoisomerase II inhibitor etoposide led to the hypothesis that exposure during pregnancy to biochemically similar compounds may promote infant acute leukemia. We established a reporter system to systematically quantitate and stratify the potential for such compounds to promote chromosomal translocations between the MLL and AF9 bcrs analogous to those in infant leukemia. We show bioflavonoids genistein and quercetin most biochemically similar to etoposide have a strong association with MLL-AF9 bcr translocations, while kaempferol, fisetin, flavone, and myricetin have a weak but consistent association, and other compounds have a minimal association in both embryonic stem (ES) and hematopoietic stem cell (HSC) populations. The frequency of translocations induced by bioflavonoids at later stages of myelopoiesis is significantly reduced by more than one log. The MLL and AF9 bcrs are sensitive to these agents and recombinogenic independent of their native context suggesting bcr sequences themselves are drivers of illegitimate DNA repair reactions and translocations, not generation of functional oncogenic fusions. This system provides for rapid systematic screening of relative risk, dose dependence, and combinatorial impact of multitudes of dietary and environmental exposures on MLL-AF9 translocations. Environ. Mol. Mutagen. 60: 154-167, 2019. © 2018 Wiley Periodicals, Inc.

Dynamic intercellular transport modulates the spatial patterning of differentiation during early neural commitment

The initiation of heterogeneity within a population of phenotypically identical progenitors is a critical event for the onset of morphogenesis and differentiation patterning. Gap junction communication within multicellular systems produces complex networks of intercellular connectivity that result in heterogeneous distributions of intracellular signaling molecules. In this study, we investigate emergent systems-level behavior of the intercellular network within embryonic stem cell (ESC) populations and corresponding spatial organization during early neural differentiation. An agent-based model incorporates experimentally-determined parameters to yield complex transport networks for delivery of pro-differentiation cues between neighboring cells, reproducing the morphogenic trajectories during retinoic acid–accelerated mouse ESC differentiation. Furthermore, the model correctly predicts the delayed differentiation and preserved spatial features of the morphogenic trajectory that occurs in response to intercellular perturbation. These findings suggest an integral role of gap junction communication in the temporal coordination of emergent patterning during early differentiation and neural commitment of pluripotent stem cells.

MSX2 Initiates and Accelerates Mesenchymal Stem/Stromal Cell Specification of hPSCs by Regulating TWIST1 and PRAME

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Human trophoblast stem cell self-renewal and differentiation: Role of decorin

The origin and regulation of stem cells sustaining trophoblast renewal in the human placenta remain unclear. Decorin, a leucine-rich proteoglycan restrains trophoblast proliferation, migration/invasiveness and endovascular differentiation, and local decorin overproduction is associated with preeclampsia (PE). Here, we tested the role of decorin in human trophoblast stem cell self-renewal and differentiation, using two models: an immortalized first trimester trophoblast cell line HTR-8/SVneo (HTR) and freshly isolated primary trophoblast (p-trophoblast) from early first trimester (6–9 weeks) placentas. Self-renewal capacity was measured by spheroid forming ability of single cells on ultra-low attachment plates for multiple generations. Markers of embryonic stem (ES) cells, trophoblast stem (TS) cells and trophoblast were used to identify stem cell hierarchy. Differentiation markers for syncytial and extravillous (EVT) pathways were employed to identify differentiated cells. Bewo cells were additionally used to explore DCN effects on syncytialization. Results reveal that the incidence of spheroid forming stem-like cells was 13–15% in HTR and 0.1–0.4%, in early first trimester p-trophoblast, including a stem cell hierarchy of two populations of ES and TS-like cells. DCN restrained ES cell self-renewal, promoted ES to TS transition and maintenance of TS cell stem-ness, but inhibited TS cell differentiation into both syncytial and EVT pathways.

Alcohol and Drug induced Neuronal Stem Cell Losses.

Kylie Shockley and Bruce ANTHONY, Department of Chemistry/Biochemistry, West Virginia Wesleyan College, Buckhannon WV, 26201.Alcohol and Drug induced Neuronal Stem Cell Losses. Alcoholism and drug addiction have become a nationwide epidemic causing substantial increases in overdose deaths and decreased quality of life. Alcohol consumption by pregnant woman leads to Fetal Alcohol Spectrum Disorders (FASD) and similar development miscues are evident from various drugs of abuse. Alcohol and drugs of abuse has been shown to reduce proliferation and increase DNA damage through both oxidative stress and alterations in cell cycle progress in both embryonic and adult stem cell populations. Cell losses from protein expressional changes at the G1/S phase checkpoint, and induces oxidative damage, thus hindering early S-phase progression through altered DNA synthesis. There is little understanding of the extent of DNA damage or the mechanism used to induce cell death. While p53 has been suspected to be a link between cell cycle and apoptotic pathways, CHK1/2 and Cdc25 could be a p53 independent connection between the damaged cell cycle and increased apoptosis seen upon alcohol and methamphetamine exposure. Research sponsored by WV-INBRE grant P20GM103434.

Asymmetricity Between Sister Cells of Pluripotent Stem Cells at the Onset of Differentiation.

Various somatic stem cells divide asymmetrically; however, it is not known whether embryonic stem cells (ESCs) divide symmetrically or asymmetrically, not only while maintaining an undifferentiated state but also at the onset of differentiation. In this study, we observed single ESCs using time-lapse imaging and compared sister cell pairs derived from the same mother cell in either the maintenance or differentiation medium. Mouse ESCs were cultured on E-cadherin-coated glass-based dishes, which allowed us to trace single cells. The undifferentiated cell state was detected by green fluorescent protein (GFP) expression driven by the Nanog promoter, which is active only in undifferentiated cells. Cell population analysis using flow cytometry showed that the peak width indicating distribution of GFP expression broadened when cells were transferred to the differentiation medium compared to when they were in the maintenance medium. This finding suggested that the population of ESCs became more heterogeneous at the onset of differentiation. Using single-cell analysis by time-lapse imaging, we found that although the total survival ratio decreased by changing to differentiation medium, the one-live-one-dead ratio of sister cell pairs was smaller compared with randomly chosen non-sister cell pairs, defined as an unsynchronized cell pair control, in both media. This result suggested that sister cell pairs were more positively synchronized with each other compared to non-sister cell pairs. The differences in interdivision time (the time interval between mother cell division and the subsequent cell division) between sister cells was smaller than that between non-sister cell pairs in both media, suggesting that sister cells divided synchronously. Although the difference in Nanog-GFP intensity between sister cells was smaller than that between non-sister cells in the maintenance medium, it was the same in differentiation medium, suggesting asymmetrical Nanog-GFP intensity. These data suggested that ESCs may divide asymmetrically at the onset of differentiation resulting in heterogeneity.

Evaluation of tools for highly variable gene discovery from single-cell RNA-seq data.

Traditional RNA sequencing (RNA-seq) allows the detection of gene expression variations between two or more cell populations through differentially expressed gene (DEG) analysis. However, genes that contribute to cell-to-cell differences are not discoverable with RNA-seq because RNA-seq samples are obtained from a mixture of cells. Single-cell RNA-seq (scRNA-seq) allows the detection of gene expression in each cell. With scRNA-seq, highly variable gene (HVG) discovery allows the detection of genes that contribute strongly to cell-to-cell variation within a homogeneous cell population, such as a population of embryonic stem cells. This analysis is implemented in many software packages. In this study, we compare seven HVG methods from six software packages, including BASiCS, Brennecke, scLVM, scran, scVEGs and Seurat. Our results demonstrate that reproducibility in HVG analysis requires a larger sample size than DEG analysis. Discrepancies between methods and potential issues in these tools are discussed and recommendations are made.

Spatial Genomic Analysis: A Multiplexed Transcriptional Profiling Method that Reveals Subpopulations of Cells Within Intact Tissues.

Here, we present Spatial Genomic Analysis (SGA), a quantitative single-cell transcriptional profiling method that takes advantage of single-molecule imaging of individual transcripts for up to a hundred genes. SGA relies on a machine learning-based image analysis pipeline that performs cell segmentation and transcript counting in a robust way. SGA is suitable for various in situ applications and was originally developed to address heterogeneity in the neural crest, which is a transient embryonic stem cell population important for formation of various vertebrate body structures. After being specified as multipotent neural crest stem cells in the dorsal neural tube, they go through an epithelial to mesenchymal transition in order to migrate to different destinations around the body, and gradually turn from stem cells to progenitors prior to final commitment. The molecular details of this process remain largely unknown, and upon their emergence, the neural crest cells have been considered as a single homogeneous population. Technical limitations have restricted the possibility to parse the neural crest cell pool into subgroups according to multiplex gene expression properties. By using SGA, we were able to identify subgroups inside the neural crest niche in the dorsal neural tube. The high sensitivity of the method allows detection of low expression levels and we were able to determine factors not previously shown to be present in neural crest stem cells, such as pluripotency or lineage markers. Finally, SGA analysis also provides prediction of gene relationships within individual cells, and thus has broad utility for powerful transcriptome analyses in original biological contexts.

Neural crest cells: Evolution, embryonic bases and craniofacial development. Systematic literature review

The neural crest cells are a population of embryonic stem cells that differentiate from the neuro-ectoderm and whose appearance constitutes an evolutionary innovation allowed vertebrates develop different morpho-functional structures to adapt from asuspensivorous eating style to a predatory style, much more efficient. This systematic literature review describes the essential role of neural crest cells in the evolutionary and embryonic development of the craniofacial structures of the vertebrates.For this purpose we searched PubMed through the Medical Subject Headings (MeSh) “neural crest” combined with the terms “neurulation, evolution, embryonic and fetal development,craniofacial development” through the Boolean connectors“AND” y “+”.
 Key words: Neurulation, neutral plata, neural crest cells, embryonic and fetal development, craniofacial development.

Electrical Bioimpedance Spectroscopy as Biosensor Technique to Identify Pluripotent Stem Cells and Fibroblasts

Pluripotent stem cells have the capacity to differentiate into specialized cells, and it has relevance in the engineering of tissue regeneration. The identification and characterization of diverse cells types requires complex techniques as flow cytometry, immunocytochemistry and the exploration of molecular markers; such techniques require infrastructure and qualified personnel. The objective of this study was to analyse the use of Electrical Bioimpedance Spectroscopy (EBIS) measurements as non-complex alternative technique to identify populations of undifferentiated pluripotent mouse Embryonic Stem Cells (mESCs) and Mouse Embryonic Fibroblasts (MEFs). EBIS measurements were compared in populations of pluripotent stem cells and fibroblasts which were characterized previously using Fourier Transform Infrared (FTIR) spectroscopy and fluorescence microscopy. The results indicate that EBIS technique has potential sensitivity at certain frequency range to discriminate between both evaluated cell populations. Additional studies with different concentrations in order to evaluate quantitatively the sensitivity and specificity of the proposed technique are recommended.

112 - Characterization and in vitro hematopoietic capacity of a very small embryonic like stem cell population isolated from human cord blood

112 - Characterization and in vitro hematopoietic capacity of a very small embryonic like stem cell population isolated from human cord blood

Tracking the embryonic stem cell transition from ground state pluripotency.

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

Concomitant differentiation of a population of mouse embryonic stem cells into neuron-like cells and schwann cell-like cells in a slow-flow microfluidic device.

To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining spiral ganglion neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF-induced mouse embryonic stem cell (mESC)-derived "neurons" could potentially substitute for lost or damaged SGN. mESC-derived "Schwann cells" produce MIF, as do all Schwann cells (Huang et al., a; Roth et al., 2007; Roth et al., 2008) and could attract SGN to a "cell-coated" implant. Neuron- and Schwann cell-like cells were produced from a common population of mESCs in an ultra-slow-flow microfluidic device. As the populations interacted, "neurons" grew over the "Schwann cell" lawn, and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF-expressing "Schwann cells" were used to coat a CI: Mouse SGN and MIF-induced "neurons" grew directionally to the CI and to a wild-type but not MIF-knockout organ of Corti explant. Two novel stem cell-based approaches for treating the problem of sensorineural hearing loss are described. Developmental Dynamics 246:7-27, 2017. © 2016 Wiley Periodicals, Inc.

Expression analysis of the endogenous Zscan4 locus and its coding proteins in mouse ES cells and preimplantation embryos

Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1–5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only ∼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.

Repression of the Aryl Hydrocarbon Receptor Is Required to Maintain Mitotic Progression and Prevent Loss of Pluripotency of Embryonic Stem Cells

Lack of cell cycle checkpoints and uninterrupted passage through S-phase continuously renew the embryonic stem (ES) cell population and maintain pluripotency. Here, we show that to regulate mitotic progression and pluripotency ES cells must keep the aryl hydrocarbon receptor (AHR), an environmental sensor and transcriptional regulator, in a persistent state of repression. This repression, however, is not always absolute, causing the AHR to fluctuate between reversible states of expression and repression, with a fraction of the cells escaping repression at any one time. Cells that escape AHR repression exhibit reduced levels of the pluripotency factors OCT4 and SOX2 and show an extended mitotic traverse time due to AHR-dependent MID1 repression and the subsequent disruption of the MID1-PP2A-CDC25B-CDK1 signaling pathway that regulates mitosis. Unlike the bulk of the cell population that differentiates into cardiomyocytes upon stimulation, AHR-expressing ES cells restrict cardiogenesis and commit to a neuroglia cell fate. It appears that the untimely expression of the Ahr gene needs to be repressed to maintain ES cell mitotic progression and prevent premature loss of pluripotency. Stem Cells 2016;34:2825-2839.