Naive Mouse Embryonic Stem Cells Research Articles

Quiescence enables unrestricted cell fate in naive embryonic stem cells

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.

Metabolic memory of Δ9-tetrahydrocannabinol exposure in pluripotent stem cells and primordial germ cells-like cells

Cannabis, the most consumed illicit psychoactive drug in the world, is increasingly used by pregnant women. However, while cannabinoid receptors are expressed in the early embryo, the impact of phytocannabinoids exposure on early embryonic processes is lacking. Here, we leverage a stepwise in vitro differentiation system that captures the early embryonic developmental cascade to investigate the impact of exposure to the most abundant phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC). We demonstrate that Δ9-THC increases the proliferation of naive mouse embryonic stem cells (ESCs) but not of their primed counterpart. Surprisingly, this increased proliferation, dependent on the CB1 receptor binding, is only associated with moderate transcriptomic changes. Instead, Δ9-THC capitalizes on ESCs’ metabolic bivalence by increasing their glycolytic rates and anabolic capabilities. A memory of this metabolic rewiring is retained throughout differentiation to Primordial Germ Cell-Like Cells in the absence of direct exposure and is associated with an alteration of their transcriptional profile. These results represent the first in-depth molecular characterization of the impact of Δ9-THC exposure on early stages of germline development.

Metabolic memory of Δ9-tetrahydrocannabinol exposure in pluripotent stem cells and primordial germ cells-like cells.

Cannabis, the most consumed illicit psychoactive drug in the world, is increasingly used by pregnant women. However, while cannabinoid receptors are expressed in the early embryo, the impact of phytocannabinoids exposure on early embryonic processes is lacking. Here, we leverage a stepwise in vitro differentiation system that captures the early embryonic developmental cascade to investigate the impact of exposure to the most abundant phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC). We demonstrate that Δ9-THC increases the proliferation of naive mouse embryonic stem cells (ESCs) but not of their primed counterpart. Surprisingly, this increased proliferation, dependent on the CB1 receptor binding, is only associated with moderate transcriptomic changes. Instead, Δ9-THC capitalizes on ESCs' metabolic bivalence by increasing their glycolytic rates and anabolic capabilities. A memory of this metabolic rewiring is retained throughout differentiation to Primordial Germ Cell-Like Cells in the absence of direct exposure and is associated with an alteration of their transcriptional profile. These results represent the first in-depth molecular characterization of the impact of Δ9-THC exposure on early stages of germline development.

A bipartite function of ESRRB can integrate signaling over time to balance self-renewal and differentiation.

Cooperative DNA binding of transcription factors (TFs) integrates the cellular context to support cell specification during development. Naive mouse embryonic stem cells are derived from early development and can sustain their pluripotent identity indefinitely. Here, we ask whether TFs associated with pluripotency evolved to directly support this state or if the state emerges from their combinatorial action. NANOG and ESRRB are key pluripotency factors that co-bind DNA. We find that when both factors are expressed, ESRRB supports pluripotency. However, when NANOG is absent, ESRRB supports a bistable culture of cells with an embryo-like primitive endoderm identity ancillary to pluripotency. The stoichiometry between NANOG and ESRRB allows quantitative titration of this differentiation, and in silico modeling of bipartite ESRRB activity suggests it safeguards plasticity in differentiation. Thus, the concerted activity of cooperative TFs can transform their effect to sustain intermediate cell identities and allow exvivo expansion of immortal stem cells. A record of this paper's transparent peer review process is included in the supplemental information.

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

A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and for modeling early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between substates has not been performed. We derived four hESC lines in naive human stem cell medium (NHSM) and generated isogenic pairs of NHSM and primed cultures. Through phenotypic, transcriptomic, and methylation profiling, we identified changes that arose during the transition to a primed substate. Although early NHSM cultures displayed naive characteristics, including greater proliferation and clonogenic potential compared with primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place human pluripotent cultures along a developmental continuum and may inform their utility for clinical and research applications.

A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression.

ABSTRACTEmbryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time.

An automated do-it-yourself system for dynamic stem cell and organoid culture in standard multi-well plates

SummaryWe present a low-cost, do-it-yourself system for complex mammalian cell culture under dynamically changing medium formulations by integrating conventional multi-well tissue culture plates with simple microfluidic control and system automation. We demonstrate the generation of complex concentration profiles, enabling the investigation of sophisticated input-response relations. We further apply our automated cell-culturing platform to the dynamic stimulation of two widely employed stem-cell-based in vitro models for early mammalian development: the conversion of naive mouse embryonic stem cells into epiblast-like cells and mouse 3D gastruloids. Performing automated medium-switch experiments, we systematically investigate cell fate commitment along the developmental trajectory toward mouse epiblast fate and examine symmetry-breaking, germ layer formation, and cardiac differentiation in mouse 3D gastruloids as a function of time-varying Wnt pathway activation. With these proof-of-principle examples, we demonstrate a highly versatile and scalable tool that can be adapted to specific research questions, experimental demands, and model systems.

TMED2 binding restricts SMO to the ER and Golgi compartments.

Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein-coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER-Golgi leading to HH signaling activation.

Slc25a36 modulates pluripotency of mouse embryonic stem cells by regulating mitochondrial function and glutathione level.

Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a36-deficient colonies under trophoblast stem cell culture conditions. As a result of Slc25a36 deficiency, mtDNA of knockdown cells declined, leading to impaired mitochondria with swollen morphology, decreased mitochondrial membrane potential, and low numbers. The key transcription regulators of mitochondrial biogenesis also decreased. These results indicate that mitochondrial dysfunction leads to an inability to support the pluripotency maintenance. Moreover, down-regulated glutathione metabolism and up-regulated focal adhesion reinforced and stabilized the process of differentiation by separately enhancing OCT4 degradation and promoting cell spread. This study improves the understanding of the function of Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.

Pluripotency on Lockdown after Deletion of Three Transcription Regulators.

Exit from the naive pluripotent state occurs through a series of changes in the gene regulatory circuitry, allowing cells to become primed for lineage commitment. In this issue of Cell Stem Cell, Kalkan etal. (2019) show that three transcription regulators are required for naive mouse embryonic stem cells (ESCs) to exit the pluripotent state.

Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency.

SummaryThe gene regulatory network (GRN) of naive mouse embryonic stem cells (ESCs) must be reconfigured to enable lineage commitment. TCF3 sanctions rewiring by suppressing components of the ESC transcription factor circuitry. However, TCF3 depletion only delays and does not prevent transition to formative pluripotency. Here, we delineate additional contributions of the ETS-family transcription factor ETV5 and the repressor RBPJ. In response to ERK signaling, ETV5 switches activity from supporting self-renewal and undergoes genome relocation linked to commissioning of enhancers activated in formative epiblast. Independent upregulation of RBPJ prevents re-expression of potent naive factors, TBX3 and NANOG, to secure exit from the naive state. Triple deletion of Etv5, Rbpj, and Tcf3 disables ESCs, such that they remain largely undifferentiated and locked in self-renewal, even in the presence of differentiation stimuli. Thus, genetic elimination of three complementary drivers of network transition stalls developmental progression, emulating environmental insulation by small-molecule inhibitors.

NODAL Secures Pluripotency upon Embryonic Stem Cell Progression fromthe Ground State.

SummaryNaive mouse embryonic stem cells (ESCs) can develop multiple fates, but the cellular and molecular processes that enable lineage competence are poorly characterized. Here, we investigated progression from the ESC ground state in defined culture. We utilized downregulation of Rex1::GFPd2 to track the loss of ESC identity. We found that cells that have newly downregulated this reporter have acquired capacity for germline induction. They can also be efficiently specified for different somatic lineages, responding more rapidly than naive cells to inductive cues. Inhibition of autocrine NODAL signaling did not alter kinetics of exit from the ESC state but compromised both germline and somatic lineage specification. Transient inhibition prior to loss of ESC identity was sufficient for this effect. Genetic ablation of Nodal reduced viability during early differentiation, consistent with defective lineage specification. These results suggest that NODAL promotes acquisition of multi-lineage competence in cells departing naive pluripotency.

A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies.

α-Ketoglutarate Accelerates the Initial Differentiation of Primed Human Pluripotent Stem Cells

Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated, primed, pluripotent states established by specific culture conditions. Increased intracellular α-ketoglutarate (αKG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of αKG or αKG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased αKG or αKG-to-succinate ratios accelerate, and elevated succinate levels delay, primed PSC differentiation. αKG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede αKG-accelerated primed PSC differentiation. Instead, αKG induced, and succinate impaired, global histone and DNA demethylation in primed PSCs. The data support αKG promotion of self-renewal or differentiation depending on the pluripotent state.

BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells.

SummaryNaive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases Tet1/2 and low levels of DNA methyltransferases Dnmt3a/b. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double Smad1;Smad5 knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation.

Generation of Naivetropic Induced Pluripotent Stem Cells from Parkinson's Disease Patients for High-Efficiency Genetic Manipulation and Disease Modeling.

The lack of robust Parkinson's disease (PD) phenotype in parkin knockout rodents and the identification of defective dopaminergic (DA) neurotransmission in midbrain DA neurons derived from induced pluripotent stem cells (iPSC) of PD patients with parkin mutations demonstrate the utility of patient-specific iPSCs as an effective system to model the unique vulnerabilities of midbrain DA neurons in PD. Significant efforts have been directed at developing efficient genomic engineering technologies in human iPSCs to study diseases such as PD. In the present study, we converted patient-specific iPSCs from the primed state to a naivetropic state by DOX-induced expression of transgenes (Oct4, Sox2, Klf4, c-Myc, and Nanog) and the use of 2iL (MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021, and human LIF). These patient-specific naivetropic iPSCs were pluripotent in terms of marker expression, spontaneous differentiation in vitro, and teratoma formation in vivo. They exhibited morphological, proliferative, and clonogenic characteristics very similar to naive mouse embryonic stem cells (ESC). The high clonal efficiency and proliferation rate of naivetropic iPSCs enabled very efficient gene targeting of GFP to the PITX3 locus by transcription activator-like effector nuclease. The naivetropic iPSCs could be readily reverted to the primed state upon the withdrawal of DOX, 2iL, and the switch to primed-state hESC culture conditions. Midbrain DA neurons differentiated from the reverted iPSCs retained the original phenotypes caused by parkin mutations, attesting to the robustness of these phenotypes and the usefulness of genomic engineering in patient-specific naivetropic iPSCs for studying PD.

In Search of Naivety

Devising a cell culture method that maintains human pluripotent stem cells (PSCs) in a state similar to naive mouse embryonic stem cells (ESCs) has been a much sought after goal in recent years. Here I consider the historical background to this quest and ask why it is considered consequential.

A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

SummaryWe describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies.

Epigenomic Comparison Reveals Activation of “Seed” Enhancers during Transition from Naive to Primed Pluripotency

Naive mouse embryonic stem cells (mESCs) and primed epiblast stem cells (mEpiSCs) represent successive snapshots of pluripotency during embryogenesis. Using transcriptomic and epigenomic mapping we show that a small fraction of transcripts are differentially expressed between mESCs and mEpiSCs and that these genes show expected changes in chromatin at their promoters and enhancers. Unexpectedly, the cis-regulatory circuitry of genes that are expressed at identical levels between these cell states also differs dramatically. In mESCs, these genes are associated with dominant proximal enhancers and dormant distal enhancers, which we term seed enhancers. In mEpiSCs, the naive-dominant enhancers are lost, and the seed enhancers take up primary transcriptional control. Seed enhancers have increased sequence conservation and show preferential usage in downstream somatic tissues, often expanding into super enhancers. We propose that seed enhancers ensure proper enhancer utilization and transcriptional fidelity as mammalian cells transition from naive pluripotency to a somatic regulatory program.

Stem cell mechanics: auxetic nuclei.

The nuclei of naive mouse embryonic stem cells that are transitioning towards differentiation expand when the cells are stretched and contract when they are compressed. What drives this auxetic phenotype is, however, unclear.