Regulation Of Embryonic Stem Cells Research Articles

RNA-Mediated Regulation of Glycolysis in Embryonic Stem Cell Pluripotency and Differentiation

Abstract Purpose of Review The maintenance and differentiation of embryonic stem cells are strictly coordinated with their metabolic status. As a core part of cellular metabolism, glycolysis provides energy and biomolecules important for stem cell proliferation and functions. Particularly, the differentiation of embryonic stem cells is associated with metabolic shifts between glycolysis and oxidative phosphorylation. However, how these metabolic swifts are regulated is still not completely known. This review aims to highlight recent advances in regulation of glycolysis at different stages of embryonic stem cells. Recent Findings Through modulating glycolysis, multiple types of RNA molecules and RNA-binding proteins are critically involved in regulation of the self-renewal, pluripotency, and differentiation of embryonic stem cells. Summary RNA-mediated glycolytic regulation in embryonic stem cells is important for their maintenance and transitions between different stages of differentiation. Better understanding of these riboregulatory mechanisms will have potential for future research and therapeutic innovation.

Rbm46 inhibits reactive oxygen species in mouse embryonic stem cells through modulating BNIP3-mediated mitophagy

Embryonic stem cells (ESCs) exhibit a metabolic preference for glycolysis over oxidative phosphorylation to meet their substantial adenosine triphosphate (ATP) demands during self-renewal. This metabolic choice inherently maintains low mitochondrial activity and minimal reactive oxygen species (ROS) generation. Nonetheless, the intricate molecular mechanisms governing the restraint of ROS production and the mitigation of cellular damage remain incompletely elucidated. In this study, we reveal the pivotal role of RNA-binding motif protein 46 (RBM46) in ESCs, acting as a direct post transcriptional regulator of ROS levels by modulating BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) mRNA expression. Rbm46 knockout lead to diminished mitochondrial autophagy, culminating in elevated ROS within ESCs, disrupting the delicate balance required for healthy self-renewal. These findings provide insights into a novel mechanism governing ROS regulation in ESCs.

Spic regulates one-carbon metabolism and histone methylation in ground-state pluripotency.

Understanding mechanisms of epigenetic regulation in embryonic stem cells (ESCs) is of fundamental importance for stem cell and developmental biology. Here, we identify Spic, a member of the ETS family of transcription factors (TFs), as a marker of ground state pluripotency. We show that Spic is rapidly induced in ground state ESCs and in response to extracellular signal-regulated kinase (ERK) inhibition. We find that SPIC binds to enhancer elements and stabilizes NANOG binding to chromatin, particularly at genes involved in choline/one-carbon (1C) metabolism such as Bhmt, Bhmt2, and Dmgdh. Gain-of-function and loss-of-function experiments revealed that Spic controls 1C metabolism and the flux of S-adenosyl methionine to S-adenosyl-L-homocysteine (SAM-to-SAH), thereby, modulating the levels of H3R17me2 and H3K4me3 histone marks in ESCs. Our findings highlight betaine-dependent 1C metabolism as a hallmark of ground state pluripotency primarily activated by SPIC. These findings underscore the role of uncharacterized auxiliary TFs in linking cellular metabolism to epigenetic regulation in ESCs.

MicroRNAs Regulate Ca2+ Homeostasis in Murine Embryonic Stem Cells.

MicroRNAs (miRNAs) are important regulators of embryonic stem cell (ESC) biology, and their study has identified key regulatory mechanisms. To find novel pathways regulated by miRNAs in ESCs, we undertook a bioinformatics analysis of gene pathways differently expressed in the absence of miRNAs due to the deletion of Dicer, which encodes an RNase that is essential for the synthesis of miRNAs. One pathway that stood out was Ca2+ signaling. Interestingly, we found that Dicer-/- ESCs had no difference in basal cytoplasmic Ca2+ levels but were hyperresponsive when Ca2+ import into the endoplasmic reticulum (ER) was blocked by thapsigargin. Remarkably, the increased Ca2+ response to thapsigargin in ESCs resulted in almost no increase in apoptosis and no differences in stress response pathways, despite the importance of miRNAs in the stress response of other cell types. The increased Ca2+ response in Dicer-/- ESCs was also observed during purinergic receptor activation, demonstrating a physiological role for the miRNA regulation of Ca2+ signaling pathways. In examining the mechanism of increased Ca2+ responsiveness to thapsigargin, neither store-operated Ca2+ entry nor Ca2+ clearance mechanisms from the cytoplasm appeared to be involved. Rather, it appeared to involve an increase in the expression of one isoform of the IP3 receptors (Itpr2). miRNA regulation of Itpr2 expression primarily appeared to be indirect, with transcriptional regulation playing a major role. Therefore, the miRNA regulation of Itpr2 expression offers a unique mechanism to regulate Ca2+ signaling pathways in the physiology of pluripotent stem cells.

DeepSea is an efficient deep-learning model for single-cell segmentation and tracking in time-lapse microscopy

Time-lapse microscopy is the only method that can directly capture the dynamics and heterogeneity of fundamental cellular processes at the single-cell level with high temporal resolution. Successful application of single-cell time-lapse microscopy requires automated segmentation and tracking of hundreds of individual cells over several time points. However, segmentation and tracking of single cells remain challenging for the analysis of time-lapse microscopy images, in particular for widely available and non-toxic imaging modalities such as phase-contrast imaging. This work presents a versatile and trainable deep-learning model, termed DeepSea, that allows for both segmentation and tracking of single cells in sequences of phase-contrast live microscopy images with higher precision than existing models. We showcase the application of DeepSea by analyzing cell size regulation in embryonic stem cells.

Orchestrating Chemical and Physical Cross-Linking in Protein Hydrogels to Regulate Embryonic Stem Cell Growth.

Protein hydrogels are ideal candidates for next-generation biomaterials due to their genetically programmable properties. Herein, we report an entirely protein-based hydrogel as an artificial extracellular matrix (ECM) for regulating the embryonic stem cell growth. A synergy between chemical and physical cross-linking was achieved in one step by SpyTag/SpyCatcher reaction and P zipper association at 37 °C. The hydrogels' stress relaxation behaviors can be tuned across a broad spectrum by single-point mutation on a P zipper. It has been found that faster relaxation can promote the growth of HeLa tumor spheroids and embryonic stem cells, and mechanical regulation of embryonic stem cells occurs via retention of the cells at the G1 phase. The results highlight the promise of genetically encoded protein materials as a platform of artificial ECM for understanding and controlling the complex cell-matrix interactions in a 3D cell culture.

INO80 Is Required for the Cell Cycle Control, Survival, and Differentiation of Mouse ESCs by Transcriptional Regulation

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation in ESCs via epigenetic mechanisms remains unclear. Here, we showed that the ATP-dependent chromatin remodeler Ino80 regulates the cell cycle genes in ESCs under primed conditions. Ino80 loss led to a significantly extended length of the G1-phase in ESCs grown under primed culture conditions. Ino80 directly bound to the transcription start site and regulated the expression of cell cycle-related genes. Furthermore, Ino80 loss induced cell apoptosis. However, the regulatory mechanism of Ino80 in differentiating ESC cycle slightly differed; an extended S-phase was detected in differentiating inducible Ino80 knockout ESCs. RNA-seq analysis of differentiating ESCs revealed that the expression of genes associated with organ development cell cycle is persistently altered in Ino80 knockout cells, suggesting that cell cycle regulation by Ino80 is not limited to undifferentiated ESCs. Therefore, our study establishes the function of Ino80 in ESC cycle via transcriptional regulation, at least partly. Moreover, this Ino80 function may be universal to other cell types.

RNA degradation eliminates developmental transcripts during murine embryonic stem cell differentiation via CAPRIN1-XRN2.

Embryonic stem cells (ESCs) are self-renewing and pluripotent. In recent years, factors that control pluripotency, mostly nuclear, have been identified. To identify non-nuclear regulators of ESCs, we screened an endogenously labeled fluorescent fusion-protein library in mouse ESCs. One of the more compelling hits was the cell-cycle-associated protein 1 (CAPRIN1). CAPRIN1 knockout had little effect in ESCs, but it significantly altered differentiation and gene expression programs. Using RIP-seq and SLAM-seq, we found that CAPRIN1 associates with, and promotes the degradation of, thousands of RNA transcripts. CAPRIN1 interactome identified XRN2 as the likely ribonuclease. Upon early ESC differentiation, XRN2 is located in the nucleus and colocalizes with CAPRIN1 in small RNA granules in a CAPRIN1-dependent manner. We propose that CAPRIN1 regulates an RNA degradation pathway operating during early ESC differentiation, thus eliminating undesired spuriously transcribed transcripts in ESCs.

Heterogeneous nuclear ribonucleoprotein U-actin complex derived from extracellular vesicles facilitates proliferation and migration of human coronary artery endothelial cells by promoting RNA polymerase II transcription

ABSTRACT Coronary artery disease (CAD) represents a fatal public threat. The involvement of extracellular vesicles (EVs) in CAD has been documented. This study explored the regulation of embryonic stem cells (ESCs)-derived EVs-hnRNPU-actin complex in human coronary artery endothelial cell (HCAEC) growth. Firstly, in vitro HCAEC hypoxia models were established. EVs were extracted from ESCs by ultracentrifugation. HCAECs were treated with EVs and si-VEGF for 24 h under hypoxia, followed by assessment of cell proliferation, apoptosis, migration, and tube formation. Uptake of EVs by HCAECs was testified. Additionally, hnRNPU, VEGF, and RNA Pol II levels were determined using Western blotting and CHIP assays. Interaction between hnRNPU and actin was evaluated by Co-immunoprecipitation assay. HCAEC viability and proliferation were lowered, apoptosis was enhanced, wound fusion was decreased, and the number of tubular capillary structures was reduced under hypoxia, whereas ESC-EVs treatment counteracted these effects. Moreover, EVs transferred hnRNPU into HCAECs. EVs-hnRNPU-actin complex increased RNA Pol II level on the VEGF gene promoter and promoted VEGF expression in HCAECs. Inhibition of hnRNPU or VEGF both annulled the promotion of EVs on HCAEC growth. Collectively, ESC-EVs-hnRNPU-actin increased RNA Pol II phosphorylation and VEGF expression, thus promoting HCAEC growth.

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Creating a United Front: Harmonizing the United States Regulatory Policies Surrounding Human Embryonic Stem Cell Research

Stem cell therapy is an imperative development in science and medicine that is heavily regulated worldwide. With the potential to cure illnesses, help understand disease development, and advance regenerative medicine, a harmonized regulatory policy is crucial to capitalize on the benefits of stem cells. This article examines an important topic of discussion surrounding stem cell therapy and research: the political debate on how and when embryonic stem cells can be used. In addition to examining ethical challenges, this article discusses the legal challenges surrounding using embryonic stem cells to inform regenerative therapies. Specifically, this article will examine the National Institute of Health’s Guidelines for Human Stem Cell Research and the historic avenues of federal and state legislation to regulate the use of these cells in research. This article discusses the internal and external inconsistencies of the United States’ current regulation of embryonic stem cells and how the divide between states is problematic for the United States’ completive stance in developmental science and medicine. Finally, this article contemplates a cohesive regulatory system influenced by individual states and other countries that currently lead the medical field, to form a united front in approaching the use of stem cells.

Role of Rho GTPases in stem cell regulation.

The future of regenerative medicine relies on our understanding of stem cells which are essential for tissue/organ generation and regeneration to maintain and/or restore tissue homeostasis. Rho family GTPases are known regulators of a wide variety of cellular processes related to cytoskeletal dynamics, polarity and gene transcription. In the last decade, major new advances have been made in understanding the regulatory role and mechanism of Rho GTPases in self-renewal, differentiation, migration, and lineage specification in tissue-specific signaling mechanisms in various stem cell types to regulate embryonic development, adult tissue homeostasis, and tissue regeneration upon stress or damage. Importantly, implication of Rho GTPases and their upstream regulators or downstream effectors in the transformation, migration, invasion and tumorigenesis of diverse cancer stem cells highlights the potential of Rho GTPase targeting in cancer therapy. In this review, we discuss recent evidence of Rho GTPase signaling in the regulation of embryonic stem cells, multiple somatic stem cells, and cancer stem cells. We propose promising areas where Rho GTPase pathways may serve as useful targets for stem cell manipulation and related future therapies.

Discovery of Klf2 interactors in mouse embryonic stem cells by immunoprecipitation-mass spectrometry utilizing exogenously expressed bait

Krüppel-like factor 2 (Klf2) is a DNA-binding transcription factor that regulates embryonic stem cell-specific gene expression. Transcription cofactors such as p300 acetyltransferase and Erk kinases interact with Klf2, providing an additional layer of transcription regulation in embryonic stem cells. To carry out a thorough survey of the Klf2 interactome in embryonic stem cells and identify novel transcription cofactors, we designed a modified immunoprecipitation-mass spectrometry (IP-MS) method. In this method, recombinant Klf2, expressed and purified from Sf9 insect cells instead of ectopically expressed in cells, was used as bait. Using this modified IP-MS method, we discovered nine Klf2-interacting proteins, including the previously reported Crebbp and p300. These proteins showed at least an 8-fold increase in signal intensity in Klf2 pull-downs compared with controls, with P-values <0.010. Among the identified Klf2-binding proteins confirmed using our IP-MS workflow was Snd1, which we found to interact directly with Klf2 and function as a transcriptional coactivator of Klf2 to drive the Oct4 gene expression. Collectively, our IP-MS protocol may offer a useful tool for identifying novel transcription cofactors in stem cells.

HnRNPLL controls pluripotency exit of embryonic stem cells by modulating alternative splicing of Tbx3 and Bptf.

The regulatory circuitry underlying embryonic stem (ES) cell self-renewal is well defined, but how this circuitry is disintegrated to enable lineage specification is unclear. RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and preliminary data suggest that they might regulate ES cell fate. By combining bioinformatic analyses with functional screening, we identified seven RBPs played important roles for the exit from pluripotency of ES cells. We characterized hnRNPLL, which mainly functions as a global regulator ofalternative splicing in ES cells. Specifically, hnRNPLL promotes multiple ES cell-preferred exon skipping events during the onset of ES cell differentiation. hnRNPLL depletion thus leads to sustained expression of ES cell-preferred isoforms, resulting in a differentiation deficiency that causes developmental defects and growth impairment in hnRNPLL-KO mice. In particular, hnRNPLL-mediated alternative splicing of two transcription factors, Bptf and Tbx3, is important for pluripotency exit. These data uncover the critical role of RBPs in pluripotency exit and suggest the application of targeting RBPs in controlling ES cell fate.

Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via β-Catenin/TCF/LEF Functions.

SummaryThe Wnt/β-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel β-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as β-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a β-catenin-dependent manner. Finally, β-catenin (ΔC) isoforms can rescue β-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.

MMTR/Dmap1 Sets the Stage for Early Lineage Commitment of Embryonic Stem Cells by Crosstalk with PcG Proteins.

Chromatin remodeling, including histone modification, chromatin (un)folding, and nucleosome remodeling, is a significant transcriptional regulation mechanism. By these epigenetic modifications, transcription factors and their regulators are recruited to the promoters of target genes, and thus gene expression is controlled through either transcriptional activation or repression. The Mat1-mediated transcriptional repressor (MMTR)/DNA methyltransferase 1 (DNMT1)-associated protein (Dmap1) is a transcription corepressor involved in chromatin remodeling, cell cycle regulation, DNA double-strand break repair, and tumor suppression. The Tip60-p400 complex proteins, including MMTR/Dmap1, interact with the oncogene Myc in embryonic stem cells (ESCs). These proteins interplay with the stem cell-related proteome networks and regulate gene expressions. However, the detailed mechanisms of their functions are unknown. Here, we show that MMTR/Dmap1, along with other Tip60-p400 complex proteins, bind the promoters of differentiation commitment genes in mouse ESCs. Hence, MMTR/Dmap1 controls gene expression alterations during differentiation. Furthermore, we propose a novel mechanism of MMTR/Dmap1 function in early stage lineage commitment of mouse ESCs by crosstalk with the polycomb group (PcG) proteins. The complex controls histone mark bivalency and transcriptional poising of commitment genes. Taken together, our comprehensive findings will help better understand the MMTR/Dmap1-mediated transcriptional regulation in ESCs and other cell types.

Pumilio proteins utilize distinct regulatory mechanisms to achieve complementary functions required for pluripotency and embryogenesis

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

SummaryCellular responses to transforming growth factor β (TGF-β) depend on cell context. Here, we explored how TGF-β/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

Genomic Integrity Safeguards Self-Renewal in Embryonic Stem Cells

SUMMARYA multitude of signals are coordinated to maintain self-renewal in embryonic stem cells (ESCs). To unravel the essential internal and external signals required for sustaining the ESC state, we expand upon a set of ESC pluripotency-associated phos-phoregulators (PRs) identified previously by short hairpin RNA (shRNA) screening. In addition to the previously described Aurka, we identify 4 additional PRs (Bub1b, Chek1, Ppm1g, and Ppp2r1b) whose depletion compromises self-renewal and leads to consequent differentiation. Global gene expression profiling and computational analyses reveal that knockdown of the 5 PRs leads to DNA damage/genome instability, activating p53 and culminating in ESC differentiation. Similarly, depletion of genome integrity-associated genes involved in DNA replication and checkpoint, mRNA processing, and Charcot-Marie-Tooth disease lead to compromise of ESC self-renewal via an increase in p53 activity. Our studies demonstrate an essential link between genomic integrity and developmental cell fate regulation in ESCs.

Autophagy and mTOR pathways in mouse embryonic stem cell, lung cancer and somatic fibroblast cell lines.

Embryonic developmental stages and regulations have always been one of the most intriguing aspects of science. Since the cancer stem cell discovery, striking for cancer development and recurrence, embryonic stem cells and control mechanisms, as well as cancer cells and cancer stem cell control mechanisms become important research materials. It is necessary to reveal the similarities and differences between somatic and cancer cells which are formed of embryonic stem cells divisions and determinations. For this purpose, mouse embryonic stem cells (mESCs), mouse skin fibroblast cells (MSFs) and mouse lung squamous cancer cells (SqLCCs) were grown in vitro and the differences between these three cell lines signalling regulations of mechanistic target of rapamycin (mTOR) and autophagic pathways were demonstrated by immunofluorescence and real-time polymerase chain reaction. Expressional differences were clearly shown between embryonic, cancer and somatic cells that mESCs displayed higher expressional level of Atg10, Hdac1 and Cln3 which are related with autophagic regulation and Hsp4, Prkca, Rhoa and ribosomal S6 genes related with mTOR activity. LC3 and mTOR protein levels were lower in mESCs than MSFs. Thus, the mechanisms of embryonic stem cell regulation results in the formation of somatic tissues whereas that these cells may be the causative agents of cancer in any deterioration.