CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.

Generation of Epiblast-Like Cells.

Different states of pluripotency can be captured in vitro depending on the embryo stage from which they are derived and the culture conditions. Pluripotency is a continuum of different states between the two extremes of naïve embryonic stem cells (ESCs) and primed Epiblast Stem Cells (EpiSCs), which resemble the pre/peri- and post- implantation embryo, respectively. The transition from naïve to primed pluripotency can be induced by growing naïve ESCs in EpiSCs medium, containing bFGF and Activin. Here we report the detailed protocol to generate and characterize the epiblast-like cells (EpiLCs), which correspond to a primed intermediate state between naïve ESCs and EpiSCs.

Generation of Gastruloids from Epiblast-Like Cells.

The different states of mouse pluripotency described so far rely on a combination of molecular, phenotypic, and functional analysis. Embryonic Stem cells (ESCs) aggregated in suspension culture are able to form 3D embryo-like structures called gastruloids that mimic features of the gastrulation process. Recent findings indicate that gastruloid formation efficiency decreases as pluripotency progresses from naïve to primed state, and suggest that gastruloids formation may represent a functional assay to discriminate different states of mouse pluripotency.Here we describe a method to generate gastruloids from Epiblast-like cells (EpiLCs), which are transiently induced from ESCs by Activin A and bFGF and represent an intermediate state from naïve ESCs to primed Epiblast Stem cells.

The Multifaceted Roles of Proline in Cell Behavior.

Herein, we review the multifaceted roles of proline in cell biology. This peculiar cyclic imino acid is: (i) A main precursor of extracellular collagens (the most abundant human proteins), antimicrobial peptides (involved in innate immunity), salivary proteins (astringency, teeth health) and cornifins (skin permeability); (ii) an energy source for pathogenic bacteria, protozoan parasites, and metastatic cancer cells, which engage in extracellular-protein degradation to invade their host; (iii) an antistress molecule (an osmolyte and chemical chaperone) helpful against various potential harms (UV radiation, drought/salinity, heavy metals, reactive oxygen species); (iv) a neural metabotoxin associated with schizophrenia; (v) a modulator of cell signaling pathways such as the amino acid stress response and extracellular signal-related kinase pathway; (vi) an epigenetic modifier able to promote DNA and histone hypermethylation; (vii) an inducer of proliferation of stem and tumor cells; and (viii) a modulator of cell morphology and migration/invasiveness. We highlight how proline metabolism impacts beneficial tissue regeneration, but also contributes to the progression of devastating pathologies such as fibrosis and metastatic cancer.

Publisher Correction: 6-Bromoindirubin-3'-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proline Metabolism in Tumor Growth and Metastatic Progression.

Cancer cells show a formidable capacity to survive under stringent conditions, to elude mechanisms of control, such as apoptosis, and to resist therapy. Cancer cells reprogram their metabolism to support uncontrolled proliferation and metastatic progression. Phenotypic and functional heterogeneity are hallmarks of cancer cells, which endow them with aggressiveness, metastatic capacity, and resistance to therapy. This heterogeneity is regulated by a variety of intrinsic and extrinsic stimuli including those from the tumor microenvironment. Increasing evidence points to a key role for the metabolism of non-essential amino acids in this complex scenario. Here we discuss the impact of proline metabolism in cancer development and progression, with particular emphasis on the enzymes involved in proline synthesis and catabolism, which are linked to pathways of energy, redox, and anaplerosis. In particular, we emphasize how proline availability influences collagen synthesis and maturation and the acquisition of cancer cell plasticity and heterogeneity. Specifically, we propose a model whereby proline availability generates a cycle based on collagen synthesis and degradation, which, in turn, influences the epigenetic landscape and tumor heterogeneity. Therapeutic strategies targeting this metabolic-epigenetic axis hold great promise for the treatment of metastatic cancers.

6-Bromoindirubin-3'-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice.

Dystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Thus, new drugs for restoring skeletal muscle deterioration are critically needed. To identify new and alternative compounds with a functional role in skeletal muscle myogenesis, we screened a library of pharmacologically active compounds and selected the small molecule 6-bromoindirubin-3'-oxime (BIO) as an inhibitor of myoblast proliferation. Using C2C12 cells, we examined BIO's effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from cell proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO is able to promote myogenic differentiation in damaged myotubes in-vitro by enriching the population of newly formed skeletal muscle myotubes. Moreover, in-vivo experiments in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers number. Additionally, we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was observed in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was observed in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was observed upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a positive modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.

Collagen Prolyl Hydroxylation-Dependent Metabolic Perturbation Governs Epigenetic Remodeling and Mesenchymal Transition in Pluripotent and Cancer Cells.

Collagen prolyl hydroxylation (CPH), which is catalyzed by prolyl 4-hydroxylase (P4H), is the most prevalent posttranslational modification in humans and requires vitamin C (VitC). Here, we demonstrate that CPH acts as an epigenetic modulator of cell plasticity. Increased CPH induced global DNA/histone methylation in pluripotent stem and tumor cells and promoted cell state transition (CST). Interfering with CPH by either genetic ablation of P4H subunit alpha-2 (P4HA2) or pharmacologic treatment reverted epigenetic changes and antagonized CST. Mechanistically, we suggest that CPH modifies the epigenetic landscape by reducing VitC for DNA and histone demethylases. Repurposed drugs targeting CPH-mediated metabolic perturbation, such as the antiasthmatic budesonide, blocked metastatic dissemination of breast cancer cells in vivo by preventing mesenchymal transition. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control CST and paves the way for the development of novel antimetastatic strategies. SIGNIFICANCE: A phenotype-based high-throughput screening reveals unforeseen metabolic control of cell plasticity and identifies budesonide as a drug candidate for metastatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3235/F1.large.jpg.

Metabolic-Epigenetic Axis in Pluripotent State Transitions.

Cell state transition (CST) occurs during embryo development and in adult life in response to different stimuli and is associated with extensive epigenetic remodeling. Beyond growth factors and signaling pathways, increasing evidence point to a crucial role of metabolic signals in this process. Indeed, since several epigenetic enzymes are sensitive to availability of specific metabolites, fluctuations in their levels may induce the epigenetic changes associated with CST. Here we analyze how fluctuations in metabolites availability influence DNA/chromatin modifications associated with pluripotent stem cell (PSC) transitions. We discuss current studies and focus on the effects of metabolites in the context of naïve to primed transition, PSC differentiation and reprogramming of somatic cells to induced pluripotent stem cells (iPSCs), analyzing their mechanism of action and the causal correlation between metabolites availability and epigenetic alteration.

Phenotype characterization of human melanoma cells resistant to dabrafenib.

In the present study, the phenotype of melanoma cells resistant to dabrafenib (a B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition. Melanoma cells resistant to dabrafenib were generated using 3 different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometric analysis, Ion Torrent technology, immunofluorescence and biochemistry. All dabrafenib-resistant cells showed, in addition to a re-activation of MAPK signaling, morphological changes compared to their sensitive counterparts, accompanied by an increase in CD90 (mesenchymal marker) expression and a decrease in E-cadherin (epithelial marker) expression, suggesting an epithelial-to-mesenchymal-like phenotypic transition. However, melanoma cells with TGF-ß1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGF­ß1­induced EMT melanoma cells, suggesting that TGF-ß1-induced EMT was not associated with dabrafenib resistance. Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/vimentin reorganization, as expected in EMT, all of them showed unvaried E-cadherin mRNA and unchanged Snail protein levels, while Twist1 protein expression was decreased with the exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected. These findings suggest a distinct active EMT-like process adopted by melanoma cells under drug exposure. Furthermore, dabrafenib-resistant cells exhibited stem cell-like features, with Oct4 translocation from the cytoplasm to peri-nuclear sites and nuclei, and increased CD20 expression. In conclusion, our data, in addition to confirming that resistance to dabrafenib is dependent on re-activation of MAPK signaling, suggest that this resistance is linked to a distinct active EMT-like process as well as stem-cell features adopted by melanoma cells.

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum.

Metabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor ß signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.

Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/ß-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.

c-Myc modulation: a key role in melanoma drug response.

Understanding molecular mechanisms involved in melanoma resistance to drugs is a big challenge. Experimental evidences suggested a correlation between mutational status in B-RAF and melanoma cell susceptibility to drugs, such as paclitaxel, doxorubicin and temozolomide, which generate an accumulation of hydrogen peroxide (H2O2) in the cells. We investigated the survival phenotype and the protein level of c-myc, a B-RAF target molecule, in melanoma cells, carrying a different mutational status in B-RAF, upon paclitaxel, doxorubicin and H2O2 treatment. For the first time, we reported c-myc modulation is critical for melanoma drug response. It appeared drug-specific and post-transcriptionally driven through PP2A; in correlation, cell pre-treatment with okadaic acid (OA), a specific PP2A inhibitor, as well as PP2A silencing of melanoma cells, was able to increase melanoma cell drug-sensitivity and c-myc protein level. This is relevant for designing efficacious therapeutic strategies in melanoma.

Nanoengineered surfaces for focal adhesion guidance trigger mesenchymal stem cell self-organization and tenogenesis.

The initial conditions for morphogenesis trigger a cascade of events that ultimately dictate structure and functions of tissues and organs. Here we report that surface nanopatterning can control the initial assembly of focal adhesions, hence guiding human mesenchymal stem cells (hMSCs) through the process of self-organization and differentiation. This process self-sustains, leading to the development of macroscopic tissues with molecular profiles and microarchitecture reminiscent of embryonic tendons. Therefore, material surfaces can be in principle engineered to set off the hMSC program toward tissuegenesis in a deterministic manner by providing adequate sets of initial environmental conditions.

L-Proline induces a mesenchymal-like invasive program in embryonic stem cells by remodeling H3K9 and H3K36 methylation.

Metabolites are emerging as key mediators of crosstalk between metabolic flux, cellular signaling, and epigenetic regulation of cell fate. We found that the nonessential amino acid L-proline (L-Pro) acts as a signaling molecule that promotes the conversion of embryonic stem cells into mesenchymal-like, spindle-shaped, highly motile, invasive pluripotent stem cells. This embryonic-stem-cell-to-mesenchymal-like transition (esMT) is accompanied by a genome-wide remodeling of the H3K9 and H3K36 methylation status. Consistently, L-Pro-induced esMT is fully reversible either after L-Pro withdrawal or by addition of ascorbic acid (vitamin C), which in turn reduces H3K9 and H3K36 methylation, promoting a mesenchymal-like-to-embryonic-stem-cell transition (MesT). These findings suggest that L-Pro, which is produced by proteolytic remodeling of the extracellular matrix, may act as a microenvironmental cue to control stem cell behavior.

An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness.

Here, we provide genetic and biochemical evidence indicating that the ability of Rhizobium etli bacteria to efficiently catabolize glutamine depends on its ability to produce reduced glutathione (l-γ-glutamyl-l-cysteinylglycine [GSH]). We find that GSH-deficient strains, namely a gshB (GSH synthetase) and a gor (GSH reductase) mutant, can use different amino acids, including histidine, alanine, and asparagine but not glutamine, as sole source of carbon, energy, and nitrogen. Moreover, l-buthionine(S,R)-sulfoximine, a GSH synthesis inhibitor, or diamide that oxidizes GSH, induced the same phenotype in the wild-type strain. Among the steps required for its utilization, glutamine uptake, occurring through the two well-characterized carriers (Aap and Bra systems) but not glutamine degradation or respiration, was largely reduced in GSH-deficient strains. Furthermore, GSH-deficient mutants of R. etli showed a reduced symbiotic efficiency. Exogenous GSH was sufficient to rescue glutamine uptake or degradation ability, as well as the symbiotic effectiveness of GSH mutants. Our results suggest a previously unknown GSH-glutamine metabolic relationship in bacteria.

An alternative succinate (2-oxoglutarate) transport system in Rhizobium tropici is induced in nodules of Phaseolus vulgaris.

The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C(4)-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.

Sulphadimethoxine inhibits Phaseolus vulgaris root growth and development of N-fixing nodules.

Sulphonamides contamination of cultivated lands occurs through the recurrent spreading of animal wastes from intensive farming. The aim of this study was to test the effect(s) of sulphadimethoxine on the beneficial N-fixing Rhizobium etli-Phaseolus vulgaris symbiosis under laboratory conditions. The consequence of increasing concentrations of sulphadimethoxine on the growth ability of free-living R. etli bacteria, as well as on seed germination, seedling development and growth of common bean plants was examined. We have established that sulphadimethoxine inhibited the growth of both symbiotic partners in a dose-dependent manner. Bacterial invasion occurring in developing root nodules was visualized by fluorescence microscopy generating EGFP-marked R. etli bacteria. Our results proved that the development of symbiotic N-fixing root nodules is hampered by sulphadimethoxine thus identifying sulphonamides as toxic compounds for the Rhizobium-legume symbiosis: a low-input sustainable agricultural practice.

Development of ectopic roots from abortive nodule primordia.

The symbiotic phenotype of five Tn5-induced mutants of Rhizobium etli affected in different anabolic pathways (namely, gluconeogenesis and biosynthesis of lysine, purine, or pyrimidine) was analyzed. These mutants induced, on the root of Phaseolus vulgaris, a normal early sequence of morphogenetics events, including root hair deformation and development of nodule primordia. Later on, however, from the resulting root outgrowths, instead of nodules, one or more ectopic roots (spaced closely related and agravitropic) emerged. Therefore, this group of mutant was collectively called "root inducer" (RIND). It was observed that the RIND-induced infection threads aborted early inside the invaded root hair, and that the resulting abortive nodules lack induction of late nodulin genes. Moreover, experiments performed using a conditional mutant (a methionine-requiring invader) revealed that bacterial invasion plays a key role in the maintenance of the program of nodule development and, in particular, in the differentiation of the most specific symbiotic tissue of globose nodules, the central tissue. These data indicate that, in P. vulgaris, the nodule primordium is a root-specified pro-meristematic tissue.