Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages.

Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.

Perchlorate and chlorate assessment in drinking water in northern Chilean cities.

Perchlorate and chlorate are endocrine disruptors considered emerging contaminants (ECs). Both oxyanions are commonly associated with anthropogenic contamination from fertilizers, pesticides, explosives, and disinfection byproducts. However, the soils of the Atacama Desert are the most extensive natural reservoirs of perchlorate in the world, compromising drinking water sources in northern Chile. Field campaigns were carried (2014-2018) to assess the presence of these ECs in the water supply networks of twelve Chilean cities. Additionally, the occurrence of perchlorate, chlorate and other anions typically observed in drinking water matrices of the Atacama Desert (i.e., nitrate, chloride, sulfate) was evaluated using a Spearman correlation analysis to determine predictors for perchlorate and chlorate. High concentrations of perchlorate (up to 114.48 µg L-1) and chlorate (up to 9650 µg L-1) were found in three northern cities. Spatial heterogeneities were observed in the physicochemical properties and anion concentrations of the water supply network. Spearman correlation analysis indicated that nitrate, chloride, and sulfate were not useful predictors for the presence of perchlorate and chlorate in drinking water in Chile. Hence, this study highlights the need to establish systematic monitoring, regulation, and treatment for these EC of drinking water sources in northern Chilean cities for public health protection.

A New Method for Sensing Soil Water Content in Green Roofs Using Plant Microbial Fuel Cells.

Green roofs have many benefits, but in countries with semiarid climates the amount of water needed for irrigation is a limiting factor for their maintenance. The use of drought-tolerant plants such as Sedum species, reduces the water requirements in the dry season, but, even so, in semiarid environments these can reach up to 60 L m-2 per day. Continuous substrate/soil water content monitoring would facilitate the efficient use of this critical resource. In this context, the use of plant microbial fuel cells (PMFCs) emerges as a suitable and more sustainable alternative for monitoring water content in green roofs in semiarid climates. In this study, bench and pilot-scale experiments using seven Sedum species showed a positive relationship between current generation and water content in the substrate. PMFC reactors with higher water content (around 27% vs. 17.5% v/v) showed larger power density (114.6 and 82.3 µW m-2 vs. 32.5 µW m-2). Moreover, a correlation coefficient of 0.95 (±0.01) between current density and water content was observed. The results of this research represent the first effort of using PMFCs as low-cost water content biosensors for green roofs.

Ectopic overexpression of Nanog induces tumorigenesis in non-tumorous fibroblasts.

Key regulatory genes in pluripotent stem cells are of interest not only as reprogramming factors but also as regulators driving tumorigenesis. Nanog is a transcription factor involved in the maintenance of embryonic stem cells and is one of the reprogramming factors along with Oct4, Sox2, and Lin28. Nanog expression has been detected in different types of tumors, and its expression is a poor prognosis for cancer patients. However, there is no clear evidence that Nanog is functionally involved in tumorigenesis. In this study, we induced overexpression of Nanog in mouse embryonic fibroblast cells and subsequently assessed their morphological changes, proliferation rate, and tumor formation ability. We found that Nanog overexpression induced immortalization of mouse embryonic fibroblast cells (MEFs) and increased their proliferation rate in vitro. We also found that formation of tumors after subcutaneous injection of retroviral-Nanog infected MEFs (N-MEFs) into athymic mouse. Cancer-related genes such as Bmi1 were expressed at high levels in N-MEFs. Hence, our results demonstrate that Nanog is able to transform normal somatic cells into tumor cells.

Human adult germline stem cells in question.

Conrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs). Here we investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues' haGSCs is therefore called into question.

Therapeutic potential of induced neural stem cells for spinal cord injury.

The spinal cord does not spontaneously regenerate, and treatment that ensures functional recovery after spinal cord injury (SCI) is still not available. Recently, fibroblasts have been directly converted into induced neural stem cells (iNSCs) by the forced expression defined transcription factors. Although directly converted iNSCs have been considered to be a cell source for clinical applications, their therapeutic potential has not yet been investigated. Here we show that iNSCs directly converted from mouse fibroblasts enhance the functional recovery of SCI animals. Engrafted iNSCs could differentiate into all neuronal lineages, including different subtypes of mature neurons. Furthermore, iNSC-derived neurons could form synapses with host neurons, thus enhancing the locomotor function recovery. A time course analysis of iNSC-treated SCI animals revealed that engrafted iNSCs effectively reduced the inflammatory response and apoptosis in the injured area. iNSC transplantation also promoted the active regeneration of the endogenous recipient environment in the absence of tumor formation. Therefore, our data suggest that directly converted iNSCs hold therapeutic potential for treatment of SCI and may thus represent a promising cell source for transplantation therapy in patients with SCI.

Synthetic greywater treatment using a scalable granular activated carbon bioelectrochemical reactor.

Greywater reuse has emerged as a promising solution for addressing water shortages. However, greywater needs treatment before reuse to meet the required water quality standards. Conventional wastewater treatment technologies are unsuitable for recreating highly decentralized domestic greywater. This study evaluated bioelectrochemical reactors (BERs) with granular activated carbon (GAC) as a sustainable alternative for developing decentralized and low-cost biological treatment systems. BERs using GAC as the anode material and conventional GAC biofilters (BFs) for synthetic greywater treatment were operated in batch mode for 110 days in two stages: (i) with polarized anodes at -150 mV vs. Ag/AgCl and (ii) as a microbial fuel cell with an external resistance of 1 kΩ. Anode polarization produced an electrosorption effect, increasing the ion removal of the BERs. Power production during the operation and cyclic voltammetry tests of the extracted granules revealed electrochemically active biofilm development on the BERs. Although low power density (0.193 ± 0.052 µW m-3) was observed in BERs, they showed a similar performance in sCOD removal (BER = 91.6-89.6 %; BF = 96.2-93.2 %) and turbidity removal (BER = 81-82 %; BF = 30-62 %) to BFs that used 50 % aeration. Additionally, scanning electron microscopy of sampled granules showed higher biomass formation in BER granules than in BF granules, suggesting a higher contribution of sessile (vs. planktonic) cells to the treatment. Thus, the results highlight the synergistic removal effect of the GAC-based BER. The scalable design presented in this study represents a proof-of-concept for developing BERs to use in decentralized greywater treatment systems.

Effect of hydrochar-doping on the performance of carbon felt as anodic electrode in microbial fuel cells.

In this study, the feasibility of using hydrochars as anodic doping materials in microbial fuel cells (MFCs) was investigated. The feedstock used for hydrochar synthesis was metal-polluted plant biomass from an abandoned mining site. The hydrochar obtained was activated by pyrolysis at 500 °C in N2 atmosphere. Under steady state conditions, the current exerted by the MFCs, as well as the cyclic voltammetry and polarization curves, showed that the activated hydrochar-doped anodes exhibited the best performance in terms of power and current density generation, 0.055 mW/cm2 and 0.15 mA/cm2, respectively. These values were approximately 30% higher than those achieved with non-doped or doped with non-activated hydrochar anodes which can be explained by the highly graphitic carbonaceous structures obtained during the hydrochar activation that reduced the internal resistance of the system. These results suggest that the activated hydrochar materials could significantly enhance the electrochemical performance of bioelectrochemical systems. Moreover, this integration will not only enhance the energy generated by MFCs, but also valorize metal polluted plant biomass within the frame of the circular economy.

Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate.

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.

Oct1 regulates trophoblast development during early mouse embryogenesis.

Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.

Concise review: challenging the pluripotency of human testis-derived ESC-like cells.

Several research groups have claimed to have successfully generated pluripotent or multipotent cells from human testis. However, the pluripotent character of those cells with respect to gene expression profile and ability to generate teratomas has been called into question. Here, we critically review these reports and provide insight to guide future studies on the derivation of human pluripotent cells from testicular tissue.

Brief report: evaluating the potential of putative pluripotent cells derived from human testis.

Human adult germline stem cells (haGSCs) were established from human testicular biopsies and were claimed to be pluripotent. Recently, the gene expression profile of haGSCs demonstrated that these cells presented with a fibroblast rather than a pluripotent identity. Nevertheless, haGSCs were reported to generate teratomas. In this report, we address this discrepancy. Instead of using haGSCs, which are no longer available for the stem cell community, we used a human testicular fibroblastic cell (hTFC) line that presents with a gene expression profile highly similar to that of haGSCs. Indeed, as shown by microarray analysis, the similarity between hTFCs and haGSCs is comparable to human embryonic stem cell (hESC) lines derived by different laboratories. We argue that the almost identical gene expression profile of hTFCs and haGSCs should result in a very similar if not identical differentiation potential. Strikingly, hTFCs were not able to generate teratomas after injection into nude mice. Instead, they formed a mesenchymal lesion that morphologically resembled the putative haGSC-derived teratomas reported previously. We conclude that haGSCs, which exhibit a profile similar to that of fibroblasts and could not generate teratomas, are not pluripotent. Future work will have to show if pluripotent cells can be derived from human testicular biopsies. Mouse work and certain testicular germ cell tumors indicate that this will be possible.

Rumen Inoculum Enhances Cathode Performance in Single-Chamber Air-Cathode Microbial Fuel Cells.

During the last decade, bioprospecting for electrochemically active bacteria has included the search for new sources of inoculum for microbial fuel cells (MFCs). However, concerning power and current production, a Geobacter-dominated mixed microbial community derived from a wastewater inoculum remains the standard. On the other hand, cathode performance is still one of the main limitations for MFCs, and the enrichment of a beneficial cathodic biofilm emerges as an alternative to increase its performance. Glucose-fed air-cathode reactors inoculated with a rumen-fluid enrichment and wastewater showed higher power densities and soluble chemical oxygen demand (sCOD) removal (Pmax = 824.5 mWm-2; ΔsCOD = 96.1%) than reactors inoculated only with wastewater (Pmax = 634.1 mWm-2; ΔsCOD = 91.7%). Identical anode but different cathode potentials suggest that differences in performance were due to the cathode. Pyrosequencing analysis showed no significant differences between the anodic community structures derived from both inocula but increased relative abundances of Azoarcus and Victivallis species in the cathodic rumen enrichment. Results suggest that this rarely used inoculum for single-chamber MFCs contributed to cathodic biofilm improvements with no anodic biofilm effects.

Bioelectrochemical chlorate reduction by Dechloromonas agitata CKB.

Chlorate has been described as an emerging pollutant that compromises water sources. In this study, bioelectrochemical reactors (BERs) using Dechloromonas agitata CKB, were evaluated as a sustainable alternative for chlorate removal. BERs were operated under flow-recirculation and batch modes with an applied cell-voltage of 0.44 V over a resistance of 1 kΩ. Results show chlorate removal up to 607.288 mg/L. After 115 days, scanning electron microscopy showed biofilm development over the electrodes, and electrochemical impedance spectroscopy confirmed the biocatalytic effect of CKB. The theoretical chlorate bioreduction potential (ε° = 0.792 V) was proven, and a kinetic study indicated that 6 electrons were involved in the reduction mechanism. Finally, a hypothetical bioelectrochemical mechanism for chlorate reduction in a BER was proposed. This research expands upon current knowledge of novel electrochemically active microorganisms and widens the scope of BER applications for chlorate removal.

Different carbon sources result in differential activation of sigma B and stress resistance in Listeria monocytogenes.

Listeria monocytogenes is an important food-borne pathogen that is ubiquitous in the environment. It is able to utilize a variety of carbon sources, to produce biofilms on food-processing surfaces and to survive food preservation-associated stresses. In this study, we investigated the effect of three common carbon sources, namely glucose, glycerol and lactose, on growth and activation of the general stress response Sigma factor, SigB, and corresponding phenotypes including stress resistance. A fluorescent reporter coupled to the promoter of lmo2230, a highly SigB-dependent gene, was used to determine SigB activation via quantitative fluorescence spectroscopy. This approach, combined with Western blotting and fluorescence microscopy, showed the highest SigB activation in lactose grown cells and lowest in glucose grown cells. In line with this observation, lactose grown cells showed the highest resistance to lethal heat and acid stress, the highest biofilm formation, and had the highest adhesion/invasion capacity in Caco-2-derived C2Bbe1 cell lines. Our data suggest that lactose utilisation triggers a strong SigB dependent stress response and this may have implications for the resistance of L. monocytogenes along the food chain.

Generation of human androgenetic induced pluripotent stem cells.

In humans, parthenogenesis and androgenesis occur naturally in mature cystic ovarian teratomas and androgenetic complete hydatidiform moles (CHM), respectively. Our previous study has reported human parthenogenetic induced pluripotent stem cells from ovarian teratoma-derived fibroblasts and screening of imprinted genes using genome-wide DNA methylation analysis. However, due to the lack of the counterparts of uniparental cells, identification of new imprinted differentially methylated regions has been limited. CHM are inherited from only the paternal genome. In this study, we generated human androgenetic induced pluripotent stem cells (AgHiPSCs) from primary androgenetic fibroblasts derived from CHM. To investigate the pluripotency state of AgHiPSCs, we analyzed their cellular and molecular characteristics. We tested the DNA methylation status of imprinted genes using bisulfite sequencing and demonstrated the androgenetic identity of AgHiPSCs. AgHiPSCs might be an attractive alternative source of human androgenetic embryonic stem cells. Furthermore, AgHiPSCs can be used in regenerative medicine, for analysis of genomic imprinting, to study imprinting-related development, and for disease modeling in humans.

Glycerol metabolism induces Listeria monocytogenes biofilm formation at the air-liquid interface.

Listeria monocytogenes is a food-borne pathogen that can grow as a biofilm on surfaces. Biofilm formation in food-processing environments is a big concern for food safety, as it can cause product contamination through the food-processing line. Although motile aerobic bacteria have been described to form biofilms at the air-liquid interface of cell cultures, to our knowledge, this type of biofilm has not been described in L. monocytogenes before. In this study we report L. monocytogenes biofilm formation at the air-liquid interface of aerobically grown cultures, and that this phenotype is specifically induced when the media is supplemented with glycerol as a carbon and energy source. Planktonic growth, metabolic activity assays and HPLC measurements of glycerol consumption over time showed that glycerol utilization in L. monocytogenes is restricted to growth under aerobic conditions. Gene expression analysis showed that genes encoding the glycerol transporter GlpF, the glycerol kinase GlpK and the glycerol 3-phosphate dehydrogenase GlpD were upregulated in the presence of oxygen, and downregulated in absence of oxygen. Additionally, motility assays revealed the induction of aerotaxis in the presence of glycerol. Our results demonstrate that the formation of biofilms at the air-liquid interface is dependent on glycerol-induced aerotaxis towards the surface of the culture, where L. monocytogenes has access to higher concentrations of oxygen, and is therefore able to utilize this compound as a carbon source.

Blockage of the Epithelial-to-Mesenchymal Transition Is Required for Embryonic Stem Cell Derivation.

Pluripotent cells emanate from the inner cell mass (ICM) of the blastocyst and when cultivated under optimal conditions immortalize as embryonic stem cells (ESCs). The fundamental mechanism underlying ESC derivation has, however, remained elusive. Recently, we have devised a highly efficient approach for establishing ESCs, through inhibition of the MEK and TGF-ß pathways. This regimen provides a platform for dissecting the molecular mechanism of ESC derivation. Via temporal gene expression analysis, we reveal key genes involved in the ICM to ESC transition. We found that DNA methyltransferases play a pivotal role in efficient ESC generation. We further observed a tight correlation between ESCs and preimplantation epiblast cell-related genes and noticed that fundamental events such as epithelial-to-mesenchymal transition blockage play a key role in launching the ESC self-renewal program. Our study provides a time course transcriptional resource highlighting the dynamics of the gene regulatory network during the ICM to ESC transition.

Molecular Obstacles to Clinical Translation of iPSCs.

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) using defined factors provides new tools for biomedical research. However, some iPSC clones display tumorigenic and immunogenic potential, thus raising concerns about their utility and safety in the clinical setting. Furthermore, variability in iPSC differentiation potential has also been described. Here we discuss whether these therapeutic obstacles are specific to transcription-factor-mediated reprogramming or inherent to every cellular reprogramming method. Finally, we address whether a better understanding of the mechanism underlying the reprogramming process might improve the fidelity of reprogramming and, therefore, the iPSC quality.

Epigenetic Aberrations Are Not Specific to Transcription Factor-Mediated Reprogramming.

Somatic cells can be reprogrammed to pluripotency using different methods. In comparison with pluripotent cells obtained through somatic nuclear transfer, induced pluripotent stem cells (iPSCs) exhibit a higher number of epigenetic errors. Furthermore, most of these abnormalities have been described to be intrinsic to the iPSC technology. Here, we investigate whether the aberrant epigenetic patterns detected in iPSCs are specific to transcription factor-mediated reprogramming. We used germline stem cells (GSCs), which are the only adult cell type that can be converted into pluripotent cells (gPSCs) under defined culture conditions, and compared GSC-derived iPSCs and gPSCs at the transcriptional and epigenetic level. Our results show that both reprogramming methods generate indistinguishable states of pluripotency. GSC-derived iPSCs and gPSCs retained similar levels of donor cell-type memory and exhibited comparable numbers of reprogramming errors. Therefore, our study demonstrates that the epigenetic abnormalities detected in iPSCs are not specific to transcription factor-mediated reprogramming.