Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells.

BACKGROUND: Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood. AIM: To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively. METHODS: mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays. RESULTS: Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression. CONCLUSION: Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.

Targeting metastasis-initiating cancer stem cells in gastric cancer with leukaemia inhibitory factor.

Gastric cancer's (GC) bad prognosis is usually associated with metastatic spread. Invasive cancer stem cells (CSC) are considered to be the seed of GC metastasis and not all CSCs are able to initiate metastasis. Targeting these aggressive metastasis-initiating CSC (MIC) is thus vital. Leukaemia inhibitory factor (LIF) is hereby used to target Hippo pathway oncogenic members, found to be induced in GC and associated with CSC features. LIF-treated GC cell lines, patient-derived xenograft (PDX) cells and/or CSC tumourspheres underwent transcriptomics, laser microdissection-associated proteomics, 2D and 3D invasion assays and in vivo xenograft in mice blood circulation. LIFR expression was analysed on tissue microarrays from GC patients and in silico from public databases. LIF-treated cells, especially CSC, presented decreased epithelial to mesenchymal transition (EMT) phenotype and invasion capacity in vitro, and lower metastasis initiation ability in vivo. These effects involved both the Hippo and Jak/Stat pathways. Finally, GC's high LIFR expression was associated with better clinical outcomes in patients. LIF treatment could thus represent a targeted anti-CSC strategy to fight against metastatic GC, and LIFR detection in primary tumours could constitute a potential new prognosis marker in this disease.

The "StemDif Sensor Test": A Straightforward, Non-Invasive Assay to Characterize the Secreted Stemness and/or Differentiation Activities of Tumor-Derived Cancer Cell Lines.

Cancer stem cells are a subpopulation of tumor cells characterized by their ability to self-renew, induce tumors upon engraftment in animals and exhibit strong resistance to chemotherapy and radiotherapy. These cells exhibit numerous characteristics in common with embryonic stem cells, expressing some of their markers, typically absent in non-pathological adult differentiated cells. The aim of this study was to investigate the potential of conditioned media from cancer stem cells to modulate the fate of Leukemia Inhibitory Factor (LIF)-dependent murine embryonic stem cells (mESCs) as a way to obtain a direct readout of the secretome of cancer cells. A functional assay, "the StemDif sensor test", was developed with two types of cancer stem cells derived from grade IV glioblastoma (adult and pediatric) or from gastric adenocarcinoma. We show that conditioned media from the selection of adult but not pediatric Glioma-Inducing Cells (GICs) maintain mESCs' pluripotency in correlation with LIF secretion and activation of STAT3 protein. In contrast, conditioned media from gastric adenocarcinoma cells display LIF-independent stemness and differentiation activities on mESC. Our test stands out for its user-friendly procedures, affordability and straightforward output, positioning it as a pioneering tool for in-depth exploration of cancer stem cell secretome characteristics.

The Journey of SCAPs (Stem Cells from Apical Papilla), from Their Native Tissue to Grafting: Impact of Oxygen Concentration.

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2.

Erratum: Rémy et al. Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties. Cells 2019, 8, 1485.

The authors wish to make the following change to their paper [...].

Leukaemia Inhibitory Factor (LIF) Inhibits Cancer Stem Cells Tumorigenic Properties through Hippo Kinases Activation in Gastric Cancer.

Cancer stem cells (CSCs) present chemo-resistance mechanisms contributing to tumour maintenance and recurrence, making their targeting of utmost importance in gastric cancer (GC) therapy. The Hippo pathway has been implicated in gastric CSC properties and was shown to be regulated by leukaemia inhibitory factor receptor (LIFR) and its ligand LIF in breast cancer. This study aimed to determine LIF's effect on CSC properties in GC cell lines and patient-derived xenograft (PDX) cells, which remains unexplored. LIF's treatment effect on CSC markers expression and tumoursphere formation was evaluated. The Hippo kinase inhibitor XMU-MP-1 and/or the JAK1 inhibitor Ruxolitinib were used to determine Hippo and canonical JAK/STAT pathway involvement in gastric CSCs' response to LIF. Results indicate that LIF decreased tumorigenic and chemo-resistant CSCs, in both GC cell lines and PDX cells. In addition, LIF increased activation of LATS1/2 Hippo kinases, thereby decreasing downstream YAP/TAZ nuclear accumulation and TEAD transcriptional activity. LIF's anti-CSC effect was reversed by XMU-MP-1 but not by Ruxolitinib treatment, highlighting the opposite effects of these two pathways downstream LIFR. In conclusion, LIF displays anti-CSC properties in GC, through Hippo kinases activation, and could in fine constitute a new CSCs-targeting strategy to help decrease relapse cases and bad prognosis in GC.

Verteporfin targeting YAP1/TAZ-TEAD transcriptional activity inhibits the tumorigenic properties of gastric cancer stem cells.

Gastric carcinomas (GC) are heterogeneous tumors, composed of a subpopulation of cluster of differentiation-44 (CD44)+ tumorigenic and chemoresistant cancer stem cells (CSC). YAP1 and TAZ oncoproteins (Y/T) interact with TEA domain family member 1 (TEAD) transcription factors to promote cell survival and proliferation in multiple tissues. Their activity and role in GC remain unclear. This work aimed to analyze Y/T-TEAD activity and molecular signature in gastric CSC, and to assess the effect of verteporfin, a Food and Drug Administration-approved drug preventing Y/T-TEAD interaction, on gastric CSC tumorigenic properties. Y/T-TEAD molecular signature was investigated using bioinformatical (KmPlot database), transcriptomic and immunostaining analyses in patient-derived GC and cell lines. Verteporfin effects on Y/T-TEAD transcriptional activity, CSC proliferation and tumorigenic properties were evaluated using in vitro tumorsphere assays and mouse models of patient-derived GC xenografts. High expressions of YAP1, TAZ, TEAD1, TEAD4 and their target genes were associated with low overall survival in nonmetastatic human GC patients (n = 444). This Y/T-TEAD molecular signature was enriched in CD44+ patient-derived GC cells and in cells resistant to conventional chemotherapy. Verteporfin treatment inhibited Y/T-TEAD transcriptional activity, cell proliferation and CD44 expression, and decreased the pool of tumorsphere-forming CD44+ /aldehyde dehydrogenase (ALDH)high gastric CSC. Finally, verteporfin treatment inhibited GC tumor growth in vivo; the residual tumor cells exhibited reduced expressions of CD44 and ALDH1, and more importantly, they were unable to initiate new tumorspheres in vitro. All these data demonstrate that Y/T-TEAD activity controls gastric CSC tumorigenic properties. The repositioning of verteporfin targeting YAP1/TAZ-TEAD activity could be a promising CSC-based strategy for the treatment of GC.

The Hippo Kinase LATS2 Controls Helicobacter pylori-Induced Epithelial-Mesenchymal Transition and Intestinal Metaplasia in Gastric Mucosa.

BACKGROUND & AIMS: Gastric carcinoma is related mostly to CagA+-Helicobacter pylori infection, which disrupts the gastric mucosa turnover and elicits an epithelial-mesenchymal transition (EMT) and preneoplastic transdifferentiation. The tumor suppressor Hippo pathway controls stem cell homeostasis; its core, constituted by the large tumor suppressor 2 (LATS2) kinase and its substrate Yes-associated protein 1 (YAP1), was investigated in this context. METHODS: Hippo, EMT, and intestinal metaplasia marker expression were investigated by transcriptomic and immunostaining analyses in human gastric AGS and MKN74 and nongastric immortalized RPE1 and HMLE epithelial cell lines challenged by H pylori, and on gastric tissues of infected patients and mice. LATS2 and YAP1 were silenced using small interfering RNAs. A transcriptional enhanced associated domain (TEAD) reporter assay was used. Cell proliferation and invasion were evaluated. RESULTS: LATS2 and YAP1 appear co-overexpressed in the infected mucosa, especially in gastritis and intestinal metaplasia. H pylori via CagA stimulates LATS2 and YAP1 in a coordinated biphasic pattern, characterized by an early transient YAP1 nuclear accumulation and stimulated YAP1/TEAD transcription, followed by nuclear LATS2 up-regulation leading to YAP1 phosphorylation and targeting for degradation. LATS2 and YAP1 reciprocally positively regulate each other's expression. Loss-of-function experiments showed that LATS2 restricts H pylori-induced EMT marker expression, invasion, and intestinal metaplasia, supporting a role of LATS2 in maintaining the epithelial phenotype of gastric cells and constraining H pylori-induced preneoplastic changes. CONCLUSIONS: H pylori infection engages a number of signaling cascades that alienate mucosa homeostasis, including the Hippo LATS2/YAP1/TEAD pathway. In the host-pathogen conflict, which generates an inflammatory environment and perturbations of the epithelial turnover and differentiation, Hippo signaling appears as a protective pathway, limiting the loss of gastric epithelial cell identity that precedes gastric carcinoma development.

Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties.

: Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O2] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.

Parabolic, Flight-Induced, Acute Hypergravity and Microgravity Effects on the Beating Rate of Human Cardiomyocytes.

Functional studies of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hCMs) under different gravity conditions contribute to aerospace medical research. To study the effects of altered gravity on hCMs, we exposed them to acute hypergravity and microgravity phases in the presence and absence of the ß-adrenoceptor isoprenalin (ISO), L-type Ca2+ channel (LTCC) agonist Bay-K8644, or LTCC blocker nifedipine, and monitored their beating rate (BR). These logistically demanding experiments were executed during the 66th Parabolic Flight Campaign of the European Space Agency. The hCM cultures were exposed to 31 alternating hypergravity, microgravity, and hypergravity phases, each lasting 20-22 s. During the parabolic flight experiment, BR and cell viability were monitored using the xCELLigence real-time cell analyzer Cardio Instrument®. Corresponding experiments were performed on the ground (1 g), using an identical set-up. Our results showed that BR continuously increased during the parabolic flight, reaching a 40% maximal increase after 15 parabolas, compared with the pre-parabolic (1 g) phase. However, in the presence of the LTCC blocker nifedipine, no change in BR was observed, even after 31 parabolas. We surmise that the parabola-mediated increase in BR was induced by the LTCC blocker. Moreover, the increase in BR induced by ISO and Bay-K8644 during the pre-parabola phase was further elevated by 20% after 25 parabolas. This additional effect reflects the positive impact of the parabolas in the absence of both agonists. Our study suggests that acute alterations of gravity significantly increase the BR of hCMs via the LTCC.

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity.

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embryonic stem cells (mESCs) to phases of hypergravity and microgravity and studied the differentiation potential of the cells using wide-genome microarray analysis. During the 64th European Space Agency's parabolic flight campaign, mESCs were exposed to 31 parabolas. Each parabola comprised phases lasting 22 s of hypergravity, microgravity, and a repeat of hypergravity. On different parabolas, RNA was isolated for microarray analysis. After exposure to 31 parabolas, mESCs (P31 mESCs) were further differentiated under normal gravity (1 g) conditions for 12 days, producing P31 12-day embryoid bodies (EBs). After analysis of the microarrays, the differentially expressed genes were analyzed using different bioinformatic tools to identify developmental and nondevelopmental biological processes affected by conditions on the parabolic flight experiment. Our results demonstrated that several genes belonging to GOs associated with cell cycle and proliferation were downregulated in undifferentiated mESCs exposed to gravity changes. However, several genes belonging to developmental processes, such as vasculature development, kidney development, skin development, and to the TGF-ß signaling pathway, were upregulated. Interestingly, similar enriched and suppressed GOs were obtained in P31 12-day EBs compared with ground control 12-day EBs. Our results show that undifferentiated mESCs exposed to alternate hypergravity and microgravity phases expressed several genes associated with developmental/differentiation and cell cycle processes, suggesting a transition from the undifferentiated pluripotent to a more differentiated stage of mESCs.

Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia.

Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF-mediated reprogramming are poorly understood. By employing a LIF-dependent 'plasticity' test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O2 despite lower levels of Pluri and Master gene expression in comparison to 20% O2.

The Oct4 protein: more than a magic stemness marker.

The Oct4 protein, encoded by the Pou5f1 gene was the very first master gene, discovered 25 years ago, to be absolutely required for the stemness properties of murine and primate embryonic stem cells. This transcription factor, which has also been shown to be essential for somatic cell reprogrammation, displays various functions depending upon its level of expression and has been quoted as a "rheostat" gene. Oct4 protein is in complexes with many different partners and its activity depends upon fine post-translational modifications. This review aims at revisiting some properties of this protein, which has not yet delivered all its potentialities.

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate.

Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF) cytokine, provide a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have led to the identification of three LIF signatures. Here we focus on muscle ras oncogene homolog (MRAS), which is a small GTPase of the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras on cell pluripotency and differentiation, we used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo, in which Mras gene structure and protein sequence are conserved. We show that persistent knockdown of Mras in mESCs reduces expression of specific master genes and that MRAS plays a crucial role in the downregulation of OCT4 and NANOG protein levels upon differentiation. In Xenopus, we demonstrate the potential of Mras to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras allows gastrula cells to retain responsiveness to fibroblast growth factor (FGF) and activin. Collectively, these results highlight novel conserved and pleiotropic effects of MRAS in stem cells and early steps of development.

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the "Functional Genomics in Embryonic Stem Cells" consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in "Expression Waves" and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.

The LIF cytokine: towards adulthood.

The aim of this article is to recapitulate the key features of leukaemia inhibitory factor cytokine (LIF), to review its numerous physiological effects and to comment on the most recent data. We will also present results of transcriptome analyses, which have highlighted different categories of LIF targets, identified in murine embryonic stem (ES) cells and early derivatives. We hope to stimulate new research fields on this puzzling cytokine, which, forty years after its discovery, has still not disclosed all its secrets.

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives.

BACKGROUND: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. RESULTS: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. CONCLUSION: We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.