• Cell differential potency
• Cell-specific Network
• Network entropy
• scRNA-seq data

• Entropy
• Cell Differentiation/genetics
• Single-Cell Analysis/methods
• Gene Expression Profiling/methods
• Sequence Analysis, RNA/methods

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths. It is often detected at a stage when there are few therapeutic options. Liver cancer stem cells (LCSCs) are highly tumorigenic and resistant to chemotherapy and radiation therapy. Their presence in HCC is a major reason why HCC is difficult to treat. The development of LCSCs is regulated by a variety of factors. This review summarizes recent advances on the factors that regulate the development of LCSCs. Due to the importance of LCSCs in the development of HCC, a better understanding of how LCSCs are regulated will help to improve the treatments for HCC patients.

• Hepatocellular carcinoma
• Liver cancer stem cells
• Pluripotency transcription factors
• Stem cell signaling
• Genetic regulators
• Epigenetic regulators

• chromatin
• epigenetics
• nuclear architecture
• phase separation
• pluripotency
• telomeres

• Wellcome Trust

• Adeno-associated virus
• Cystic fibrosis
• Lung airway mucus
• Recombinant capsids
• Stem cells
• Tissue-specific promoters

Dental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity. N⁶-methyladenosine (m⁶A) is the most prevalent, reversible internal modification in RNAs associated with stem cell fate determination. In this study, we aim to explore the biological effect of m⁶A methylation in DPSCs.

m⁶A immunoprecipitation with deep sequencing (m⁶A RIP-seq) demonstrated the features of m⁶A modifications in DPSC transcriptome. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence, and apoptosis were analyzed by β-galactosidase, TUNEL staining, and flow cytometry. Bioinformatic analysis combing m⁶A RIP and shMETTL3 RNA-seq functionally enriched overlapped genes and screened target of METTL3. Cell cycle distributions were assayed by flow cytometry, and m⁶A RIP-qPCR was used to confirm METTL3-mediated m⁶A methylation.

Here, m⁶A peak distribution, binding area, and motif in DPSCs were first revealed by m⁶A RIP-seq. We also found a relatively high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. A conjoint analysis of m⁶A RIP and RNA sequencing showed METTL3 depletion associated with cell cycle, mitosis, and alteration of METTL3 resulted in cell cycle arrest. Furthermore, the protein interaction network of differentially expressed genes identified Polo-like kinase 1 (PLK1), a critical cycle modulator, as the target of METTL3-mediated m⁶A methylation in DPSCs.

These results revealed m⁶A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and served new insight into stem cell-based tissue engineering.

The online version contains supplementary material available at 10.1186/s13287-021-02223-x.

• Adult stem cells
• Cell apoptosis
• Cell cycle
• Dental pulp
• RNA epigenetics

It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial–mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

During early development, extrinsic triggers prompt pluripotent cells to begin the process of differentiation. When and how human embryonic stem cells (hESCs) irreversibly commit to differentiation is a fundamental yet unanswered question. By combining single-cell imaging, genomic approaches, and mathematical modeling, we find that hESCs commit to exiting pluripotency unexpectedly early. We show that bone morphogenetic protein 4 (BMP4), an important differentiation trigger, induces a subset of early genes to mirror the sustained, bistable dynamics of upstream signaling. Induction of one of these genes, GATA3, drives differentiation in the absence of BMP4. Conversely, GATA3 knockout delays differentiation and prevents fast commitment to differentiation. We show that positive feedback at the level of the GATA3-BMP4 axis induces fast, irreversible commitment to differentiation. We propose that early commitment may be a feature of BMP-driven fate choices and that interlinked feedback is the molecular basis for an irreversible transition from pluripotency to differentiation.

•

Irreversible commitment to BMP4-driven hESC differentiation is fast

• BMP4
• GATA3
• bistability
• commitment
• differentiation
• fate decisions
• hESC
• positive feedback

The consumption of omega 3 polyunsaturated fatty acids (PUFAs) is important for human health and is closely associated with cell proliferation and differentiation. This study aimed to investigate the influence of omega 3 PUFAs on embryonic stem cell (ESC) proliferation and explore potential mechanisms that mediate these effects.

In this study, we isolated ESCs from fad3b-expressing transgenic mice. We detected the fatty-acid composition of ESCs using gas chromatography-mass spectroscopy, analyzed cell-cycle phases using flow cytometry, and detected gene expression using real-time polymerase chain reaction (PCR) and western blots.

The amount of omega 3 PUFAs significantly increased in fad3b versus control ESCs. However, the growth of fad3b ESCs was slower than that of control cells, and most fad3b ESCs were in a prolonged G0/G1 phase after being passaged for 18 h. Therefore, we hypothesized that fad3b expression inhibited the cell cycle in ESCs by increasing the expression of P21, which then decreased the expression of cyclin-dependent kinase 4 (Cdk4). We found that pretreatment of fad3b ESCs with PD0325901, a P21 inhibitor, clearly attenuated the inhibitory effects of P21 on Cdk4, and resumed the cell cycle.

Expression of the fad3b gene in ESCs increased the omega 3 PUFA content, which inhibited cell proliferation by prolonging the G1 phase but did not arrest the G0-to-G1 or G1-to-S transitions. The prolonged G1 phase in fad3b ESCs was probably induced by downregulation of Cdk4 expression via p21 upregulation. These results suggest that accumulation of omega 3 PUFAs in vivo may beneficially affect ESC differentiation and that fad3b ESCs may be a useful tool for investigating related mechanisms.

The online version of this article (10.1186/s12944-018-0862-x) contains supplementary material, which is available to authorized users.

• Cdk4
• Cell cycle
• Embryonic stem cells
• P21
• fad3b

Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of in vitro and in vivo PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both in vitro and in vivo screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation.

• PDD
• mouse
• phenotype
• small molecules
• stem cells
• zebrafish

The coordinated development of the nervous system requires fidelity in the expression of specific genes determining the different neural cell phenotypes. Stem cell fate decisions during neurodevelopment are strictly correlated with their epigenetic status. The epigenetic regulatory processes, such as DNA methylation and histone modifications discussed in this review article, may impact both neural stem cell (NSC) self-renewal and differentiation and thus play an important role in neurodevelopment. At the same time, stem cell decisions regarding fate commitment and differentiation are highly dependent on the temporospatial expression of specific genes contingent on the developmental stage of the nervous system. An interplay between the above, as well as basic cell processes, such as transcription regulation, DNA replication, cell cycle regulation and DNA repair therefore determine the accuracy and function of neuronal connections. This may significantly impact embryonic health and development as well as cognitive processes such as neuroplasticity and memory formation later in the adult.

• acetylation
• epigenetics
• methylation
• neurodevelopment
• neuroplasticity
• stem cells

• Dclk1
• pluripotency
• quiescence
• self-renewal

• Cell Differentiation
• Cytomegalovirus/physiology
• Cytomegalovirus Infections/pathology
• Cytomegalovirus Infections/virology
• Female
• Humans
• Placenta/virology
• Pregnancy
• Pregnancy Complications, Infectious/pathology
• Pregnancy Complications, Infectious/virology
• Stem Cells/physiology
• Stem Cells/virology
• Trophoblasts/physiology
• Trophoblasts/virology
• Virus Replication

• R21 HD061890 NICHD NIH HHS
• R44AI102396 NIAID NIH HHS
• R01 AI046657 NIAID NIH HHS
• R44 AI102396 NIAID NIH HHS
• R56 AI101130 NIAID NIH HHS
• R01AI046657 NIAID NIH HHS
• R56AI101130 NIAID NIH HHS

Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen.

• Embryonic stem cells
• Neural differentiation
• Primary cilium

• embryonic stem cells
• pluripotency
• cell cycle

• Animals
• Cell Cycle/physiology
• Cell Nucleus/physiology
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/physiology
• Gene Expression Regulation/physiology
• Genes, myc/physiology
• Histones/genetics
• Histones/metabolism
• Humans
• MicroRNAs
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/physiology

• P01 AR048818 NIAMS NIH HHS
• RC1 AG035886 NIA NIH HHS
• R01 AR039588 NIAMS NIH HHS
• AR48818 NIAMS NIH HHS
• CA139322 NCI NIH HHS
• CA82834 NCI NIH HHS
• P01 CA082834 NCI NIH HHS
• AR039588 NIAMS NIH HHS
• R01 CA139322 NCI NIH HHS
• AG035886 NIA NIH HHS
• R01 AR049069 NIAMS NIH HHS

• Animals
• Cell Cycle/genetics
• Cell Cycle/physiology
• Cell Cycle Proteins/genetics
• Cell Cycle Proteins/metabolism
• Cell Line
• Chromatin Immunoprecipitation
• Mice
• Models, Theoretical
• Oligonucleotide Array Sequence Analysis
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/metabolism
• Trans-Activators/genetics
• Trans-Activators/metabolism

• Intramural NIH HHS

• chromatin state
• hox
• myogenesis
• polycomb
• quiescence
• regeneration
• satellite cells
• trithorax

• DNA damage
• OCT4A/POU5F1
• TP53
• pluripotency
• self-renewal
• senescence
• tumor cells

• apoptosis
• cell cycle
• differentiation
• embryonic stem cell
• p53
• nucleolin
• self-renewal

• Animals
• Blotting, Western
• Cell Cycle/genetics
• Cell Cycle/physiology
• Cell Differentiation/genetics
• Cell Differentiation/physiology
• Cell Line
• Cell Proliferation
• Embryoid Bodies/cytology
• Embryoid Bodies/metabolism
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/metabolism
• Mice
• NIH 3T3 Cells
• Oligonucleotide Array Sequence Analysis
• Phosphoproteins/genetics
• Phosphoproteins/metabolism
• RNA, Small Interfering/genetics
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Real-Time Polymerase Chain Reaction
• Tumor Suppressor Protein p53/genetics
• Tumor Suppressor Protein p53/metabolism
• Nucleolin

• differentiation
• intestine
• micro-rna
• crypt-luminal axis

The RNA binding protein, DEAD END (DND1), is essential for maintaining viable germ cells in vertebrates. It is also a testicular germ cell tumor susceptibility factor in mice. DND1 has been shown to interact with the 3'-untranslated region (3'-UTR) of mRNAs such as P27 and LATS2. Binding of DND1 to the 3'-UTRs of these transcripts blocks the inhibitory function of microRNAs (miRNA) from these transcripts and in this way DND1 helps maintain P27 and LATS2 protein expression. We found that DND1 is also expressed in embryonic stem (ES) cells. Because ES cells share similar gene expression patterns as germ cells, we utilized ES cells to identify additional candidate mRNAs that associate with DND1.

ES cells are readily amenable to genetic modification and easier to culture in vitro compared to germ cells. Therefore, for the purpose of our study, we made a genetically modified, stable, human embryonic stem (hES) cell line that expresses hemagluttinin (HA)-tagged DND1 in a doxycycline (dox) regulatable manner. This line expresses modest levels of HA-DND1 and serves as a good system to study DND1 function in vitro. We used this stable cell line to identify the transcripts that physically interact with DND1. By performing ribonucleoprotein immunoprecipitation (RIP) followed by RT-PCR, we identified that transcripts encoding pluripotency factors (OCT4, SOX2, NANOG, LIN28), cell cycle regulators (TP53, LATS2) and apoptotic factors (BCLX, BAX) are specifically associated with the HA-DND1 ribonucleoprotein complex. Surprisingly, in many cases, bioinformatics analysis of the pulled-down transcripts did not reveal the presence of known DND1 interacting motifs.

Our results indicate that the inducible ES cell line system serves as a suitable in vitro system to identify the mRNA targets of DND1. The RIP-RT results hint at the broad spectrum of mRNA targets that interact with DND1 in ES cells. Based on what is known about DND1 function, our results suggest that DND1 may impose another level of translational regulation to modulate expression of critical factors in ES cells.

Excessive cell growth in Drosophila intestinal stem cells lacking TSC blocks further cell division.

Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. We demonstrate in this paper that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, we report in this paper that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs expressed proper stem cell markers, did not differentiate, and had defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc was sufficient to rescue the division defect. The TSC mutant guts had a thinner epithelial structure than wild-type tissues, and the mutant flies were more susceptible to tissue damage. Therefore, we have uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division.

• mybl2
• differentiation
• colon
• commitment
• cell cycle

• Base Sequence
• Caco-2 Cells
• Cell Cycle Proteins/genetics
• Cell Cycle Proteins/metabolism
• Cell Differentiation/genetics
• Cell Proliferation
• Colon/cytology
• Epithelial Cells/cytology
• Epithelial Cells/metabolism
• G2 Phase/genetics
• Gene Expression Regulation
• Gene Knockdown Techniques
• Humans
• Mitosis/genetics
• Models, Biological
• Protein Binding
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• RNA, Small Interfering/metabolism
• Trans-Activators/genetics
• Trans-Activators/metabolism
• Transcription Initiation Site
• Transcription, Genetic
• Transfection

• CA114265 NCI NIH HHS
• CA123473 NCI NIH HHS
• R21 CA123473 NCI NIH HHS
• P013330 PHS HHS
• P30 CA013330 NCI NIH HHS
• R01 CA114265 NCI NIH HHS
• R21 CA137508 NCI NIH HHS
• U54 CA100926 NCI NIH HHS

Embryonic stem (ES) and induced-pluripotent stem (iPS) cells can be grown indefinitely under appropriate conditions whilst retaining the ability to differentiate to cells representative of the three primary germ layers. Such cells have the potential to revolutionize medicine by offering treatment options for a wide range of diseases and disorders as well as providing a model system for elucidating mechanisms involved in development and disease. In recent years, evidence for the function of E-cadherin in regulating pluripotent and self-renewal signaling pathways in ES and iPS cells has emerged. In this review, we discuss the function of E-cadherin and its interacting partners in the context of development and disease. We then describe relevant literature highlighting the function of E-cadherin in establishing and maintaining pluripotent and self-renewal properties of ES and iPS cells. In addition, we present experimental data demonstrating that exposure of human ES cells to the E-cadherin neutralizing antibody SHE78.7 allows culture of these cells in the absence of FGF2-supplemented medium.

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.

Stem cells have received considerable public attention in part because of their potential application in regenerative therapies. Stem cells can be operationally defined as cells that have the unique property to self-renew, as well as to generate more differentiated progeny (differentiation). However, much remains to be learned about the genes regulating stem cell differentiation and renewal, their relationship to each other, and the signaling pathways that control their expression and/or activity. In this paper, we present a new resource developed in our laboratory, called DelES, for chromosomal deletion in ES cells. By reinserting deleted DNA fragments in a set of ESC clones harboring nested chromosomal deletions, we identified the Rps14 gene as being haploinsufficient for embryoid body formation. We think that our library of more than 1,300 clones represents a new resource that should allow the identification of genes and other elements that are essential for stem cell activity.

Cellular senescence involves a reduction in adult stem cell self-renewal, and epigenetic regulation of gene expression is one of the main underlying mechanisms. Here, we observed that the cellular senescence of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) caused by inhibition of histone deacetylase (HDAC) activity leads to down-regulation of high mobility group A2 (HMGA2) and, on the contrary, to up-regulation of p16^INK4A, p21^CIP1/WAF1 and p27^KIP1. We found that let-7a1, let-7d, let-7f1, miR-23a, miR-26a and miR-30a were increased during replicative and HDAC inhibitor-mediated senescence of hUCB-MSCs by microRNA microarray and real-time quantitative PCR. Furthermore, the configurations of chromatins beading on these miRNAs were prone to transcriptional activation during HDAC inhibitor-mediated senescence. We confirmed that miR-23a, miR-26a and miR-30a inhibit HMGA2 to accelerate the progress of senescence. These findings suggest that HDACs may play important roles in cellular senescence by regulating the expression of miRNAs that target HMGA2 through histone modification.

• induced pluripotent (ips) stem cells
• embryonic stem cells
• development and disease
• vasculature
• heart

• Aging
• Animals
• Blood Vessels/metabolism
• Blood Vessels/pathology
• Cardiovascular Diseases/pathology
• Cardiovascular Diseases/physiopathology
• Cardiovascular Diseases/therapy
• Embryonic Stem Cells/metabolism
• Embryonic Stem Cells/pathology
• Guided Tissue Regeneration
• Humans
• Induced Pluripotent Stem Cells/metabolism
• Induced Pluripotent Stem Cells/pathology
• Myocardium/metabolism
• Myocardium/pathology
• Stem Cell Transplantation

• Z01 AG000288 Intramural NIH HHS