Principles of signaling pathway modulation for enhancing human naive pluripotency induction.

Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/ß-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

Establishing the human naïve pluripotent state.

Pluripotency is first assembled within the inner-cell-mass of developing pre-implantation blastocysts, and is gradually reconfigured and dismantled during early post-implantation development, before overt differentiation into somatic lineages ensues. This transition from pre-implantation to post-implantation pluripotent states, respectively referred to as naïve and primed, is accompanied by dramatic changes in molecular and functional characteristics. Remarkably, pluripotent states can be artificially preserved in a self-renewing state in vitro by continuous supplementation of a variety of exogenous cytokines and small molecule inhibitors. Different exogenous factors endow the cells with distinct configurations of pluripotency that have direct influence on stem cell characteristics both in mice and humans. Here we overview pluripotent states captured from rodents and humans under different growth conditions, and provide a conceptual framework for classifying pluripotent cell states on the basis of a combination of multiple characteristics that a pluripotent cell can simultaneously retain. We further highlight the complexity and dynamic nature of these artificially isolated in vitro pluripotent states in humans.

Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

SOX17 is a critical specifier of human primordial germ cell fate.

Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

Derivation of novel human ground state naive pluripotent stem cells.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3ß signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Widespread promoter-mediated coordination of transcription and mRNA degradation.

BACKGROUND: Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. RESULTS: We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. CONCLUSIONS: Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation.

Chromosomal duplication is a transient evolutionary solution to stress.

Aneuploidy, an abnormal number of chromosomes, is a widespread phenomenon found in unicellulars such as yeast, as well as in plants and in mammalians, especially in cancer. Aneuploidy is a genome-scale aberration that imposes a severe burden on the cell, yet under stressful conditions specific aneuploidies confer a selective advantage. This dual nature of aneuploidy raises the question of whether it can serve as a stable and sustainable evolutionary adaptation. To clarify this, we conducted a set of laboratory evolution experiments in yeast and followed the long-term dynamics of aneuploidy under diverse conditions. Here we show that chromosomal duplications are first acquired as a crude solution to stress, yet only as transient solutions that are eliminated and replaced by more efficient solutions obtained at the individual gene level. These transient dynamics of aneuploidy were repeatedly observed in our laboratory evolution experiments; chromosomal duplications gained under stress were eliminated not only when the stress was relieved, but even if it persisted. Furthermore, when stress was applied gradually rather than abruptly, alternative solutions appear to have emerged, but not aneuploidy. Our findings indicate that chromosomal duplication is a first evolutionary line of defense, that retains survivability under strong and abrupt selective pressures, yet it merely serves as a "quick fix," whereas more refined and sustainable solutions take over. Thus, in the perspective of genome evolution trajectory, aneuploidy is a useful yet short-lived intermediate that facilitates further adaptation.

Long-term monitoring of age-related macular degeneration with preferential hyperacuity perimetry.

BACKGROUND AND OBJECTIVE: To estimate the efficacy and usability of preferential hyper-acuity perimetry (PHP) for monitoring patients with high-risk intermediate age-related macular degeneration (AMD). PATIENTS AND METHODS: A long-term, observational, prospective case series of patients with intermediate AMD who underwent fluorescein angiography at recruitment. Eyes were examined every 3 months with PHP, visual acuity, and biomicroscopy. Optical coherence tomography (OCT) imaging was performed when PHP was outside normal limits. In case of suspected findings in OCT, fluorescein angiography was also performed. Patients diagnosed as having choroidal neovascularization (CNV) were offered anti-vascular endothelial growth factor therapy. RESULTS: Twenty-six eyes (25 patients) were monitored for a mean follow-up period of 600 days. Of the 172 PHP tests done by these 26 eyes with intermediate AMD, 158 were within normal limits yielding a false-positive rate of 8.1%. Three of 4 eyes that converted to CNV had PHP test results outside normal limits before or on the day of diagnosis. CONCLUSION: PHP is useful for detecting CNV in regularly monitored eyes with intermediate AMD while maintaining a low false-positive rate.

Toward earlier detection of choroidal neovascularization secondary to age-related macular degeneration: multicenter evaluation of a preferential hyperacuity perimeter designed as a home device.

PURPOSE: The primary purpose of this study was to evaluate the ability of a home device preferential hyperacuity perimeter to discriminate between patients with choroidal neovascularization (CNV) and intermediate age-related macular degeneration (AMD), and the secondary purpose was to investigate the dependence of sensitivity on lesion characteristics. METHODS: All participants were tested with the home device in an unsupervised mode. The first part of this work was retrospective using tests performed by patients with intermediate AMD and newly diagnosed CNV. In the second part, the classifier was prospectively challenged with tests performed by patients with intermediate AMD and newly diagnosed CNV. The dependence of sensitivity on lesion characteristics was estimated with tests performed by patients with CNV of both parts. RESULTS: In 66 eyes with CNV and 65 eyes with intermediate AMD, both sensitivity and specificity were 0.85. In the retrospective part (34 CNV and 43 intermediate AMD), sensitivity and specificity were 0.85 +/- 0.12 (95% confidence interval) and 0.84 +/- 0.11 (95% confidence interval), respectively. In the prospective part (32 CNV and 22 intermediate AMD), sensitivity and specificity were 0.84 +/- 0.13 (95% confidence interval) and 0.86 +/- 0.14 (95% confidence interval), respectively. Chi-square analysis showed no dependence of sensitivity on type (P = 0.44), location (P = 0.243), or size (P = 0.73) of the CNV lesions. CONCLUSION: A home device preferential hyperacuity perimeter has good sensitivity and specificity in discriminating between patients with newly diagnosed CNV and intermediate AMD. Sensitivity is not dependent on lesion characteristics.

Synaptic depression in conjunction with A-current channels promote phase constancy in a rhythmic network.

In many central pattern generators, pairs of neurons maintain an approximately fixed phase despite large changes in the frequency. The mechanisms underlying phase maintenance are not clear. Previous theoretical work suggested that inhibitory synapses that show short-term depression could play a critical role in this respect. In this work we examine how the interaction between synaptic depression and the kinetics of a transient potassium (A-like) current could be advantageous for phase constancy in a rhythmic network. To demonstrate the mechanism in the context of a realistic central pattern generator, we constructed a detailed model of the crustacean pyloric circuit. The frequency of the rhythm was modified by changing the level of a ligand-activated current in one of the pyloric neurons. We examined how the time difference of firing activities between two selected neurons in this circuit is affected by synaptic depression, A-current, and a combination of the two. We tuned the parameters of the model such that with synaptic depression alone, or A-current alone, phase was not maintained between these two neurons. However, when these two components came together, they acted synergistically to maintain the phase across a wide range of cycle periods. This suggests that synaptic depression may be necessary to allow an A-current to delay a postsynaptic neuron in a frequency-dependent manner, such that phase invariance is ensured.

Intercircuit control via rhythmic regulation of projection neuron activity.

Synaptic feedback from rhythmically active neuronal circuits commonly causes their descending inputs to exhibit the rhythmic activity pattern generated by that circuit. In most cases, however, the function of this rhythmic feedback is unknown. In fact, generally these inputs can still activate the target circuit when driven in a tonic activity pattern. We are using the crab stomatogastric nervous system (STNS) to test the hypothesis that the neuronal circuit-mediated rhythmic activity pattern in projection neurons contributes to intercircuit regulation. The crab STNS contains an identified projection neuron, modulatory commissural neuron 1 (MCN1), whose tonic stimulation activates and modulates the gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the stomatogastric ganglion (STG). During tonic stimulation of MCN1, the pyloric circuit regulates both gastric mill cycle frequency and gastropyloric coordination via a direct synapse onto a gastric mill neuron in the STG. However, when MCN1 is spontaneously active, it has a pyloric-timed activity pattern attributable to synaptic input from the pyloric circuit. This pyloric-timed activity in MCN1 provides the pyloric circuit with a second pathway for regulating the gastric mill rhythm. At these times, the direct STG synapse from the pyloric circuit to the gastric mill circuit is not necessary for pyloric regulation of the gastric mill rhythm. However, in the intact system, these two pathways play complementary roles in this intercircuit regulation. Thus, one role for rhythmicity in modulatory projection neurons is to enable them to mediate the interactions between distinct but related neuronal circuits.

The activity phase of postsynaptic neurons in a simplified rhythmic network.

Many inhibitory rhythmic networks produce activity in a range of frequencies. The relative phase of activity between neurons in these networks is often a determinant of the network output. This relative phase is determined by the interaction between synaptic inputs to the neurons and their intrinsic properties. We show, in a simplified network consisting of an oscillator inhibiting a follower neuron, how the interaction between synaptic depression and a transient potassium current in the follower neuron determines the activity phase of this neuron. We derive a mathematical expression to determine at what phase of the oscillation the follower neuron becomes active. This expression can be used to understand which parameters determine the phase of activity of the follower as the frequency of the oscillator is changed. We show that in the presence of synaptic depression, there can be three distinct frequency intervals, in which the phase of the follower neuron is determined by different sets of parameters. Alternatively, when the synapse is not depressing, only one set of parameters determines the phase of activity at all frequencies.

Short-term dynamics of a mixed chemical and electrical synapse in a rhythmic network.

In the rhythmically active pyloric circuit of the spiny lobster, the synapse between the lateral pyloric (LP) neuron and pyloric constrictor (PY) neuron has an inhibitory depressing chemical and an electrical component. To understand how the dynamics of the LP-->PY synapse affect the relative firing times between these two neurons in an ongoing rhythm, we characterized the dynamics of the LP-->PY synapse after a pharmacological block of ongoing activity. When a train of voltage pulses was applied to the voltage-clamped LP neuron, the inhibitory chemical component of the postsynaptic potential (PSP) in the PY neuron rapidly depressed. Thus, after the first few pulses, the PSP was either hyperpolarizing or depolarizing, depending on the interpulse duration, with shorter interpulse durations producing depolarizing PSPs. To characterize the synaptic response during rhythmic activity, we played back prerecorded realistic waveforms in the voltage-clamped LP neuron. After an initial transient, the resulting PSP in PY was always depolarizing, suggesting that in an ongoing rhythm, the electrical component of the synapse is dominant. However, our results indicate that the chemical component of the synapse acts to delay the peak time of the PSP and to reduce its amplitude, and that these effects become more important at slower cycle periods.

Contribution of synaptic depression to phase maintenance in a model rhythmic network.

In many rhythmic neuronal networks that operate in a wide range of frequencies, the time of neuronal firing relative to the cycle period (the phase) is invariant. This invariance suggests that when frequency changes, firing time is precisely adjusted either by intrinsic or synaptic mechanisms. We study the maintenance of phase in a computational model in which an oscillator neuron (O) inhibits a follower neuron (F) by comparing the dependency of phase on cycle period in two cases: when the inhibitory synapse is depressing and when it is nondepressing. Of the numerous ways of changing the cycle period, we focus on three cases where either the duration of the active state, the inactive state, or the duty cycle of neuron O remains constant. In each case, we measure the phase at which neuron F fires with respect to the onset of firing in neuron O. With a nondepressing synapse, this phase is generally a monotonic function of cycle period except in a small parameter range in the case of the constant inactive duration. In contrast, with a depressing synapse, there is always a parameter regime in which phase is a cubic function of cycle period: it decreases at short cycle periods, increases in an intermediate range, and decreases at long cycle periods. This complex shape for the phase-period relationship arises because of the interaction between synaptic dynamics and intrinsic properties of the postsynaptic neuron. By choosing appropriate parameters, the cubic shape of the phase-period curve results in a small variation in phase for a large interval of periods. Consequently, we find that although a depressing synapse does not produce perfect phase maintenance, in most cases it is superior to a nondepressing synapse in promoting a constant phase difference.