Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle.

The induction of the germ cell lineage from pluripotent stem cells (in vitro gametogenesis) will help understand the mechanisms underlying germ cell differentiation and provide an alternative source of gametes for reproduction. This technology is especially important for cattle, which are among the most important livestock species for milk and meat production. Here, we developed a new method for robust induction of primordial germ cell-like cells (PGCLCs) from newly established bovine embryonic stem (bES) cells. First, we refined the pluripotent culture conditions for pre-implantation embryos and ES cells. Inhibition of RHO increased the number of epiblast cells in the pre-implantation embryos and dramatically improved the efficiency of ES cell establishment. We then determined suitable culture conditions for PGCLC differentiation using bES cells harboring BLIMP1-tdTomato and TFAP2C-mNeonGreen (BTTN) reporter constructs. After a 24-h culture with bone morphogenetic protein 4 (BMP4), followed by three-dimensional culture with BMP4 and a chemical agonist and WNT signaling chemical antagonist, bES cells became positive for the reporters. A set of primordial germ cells (PGC) marker genes, including PRDM1/BLIMP1, TFAP2C, SOX17, and NANOS3, were expressed in BTTN-positive cells. These bovine PGCLCs (bPGCLCs) were isolated as KIT/CD117-positive and CD44-negative cell populations. We anticipate that this method for the efficient establishment of bES cells and induction of PGCLCs will be useful for stem cell-based reproductive technologies in cattle.

A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals.

Differential speeds in biochemical reactions have been proposed to be responsible for the differences in developmental tempo between mice and humans. However, the underlying mechanism controlling the species-specific kinetics remains to be determined. Using in vitro differentiation of pluripotent stem cells, we recapitulated the segmentation clocks of diverse mammalian species varying in body weight and taxa: marmoset, rabbit, cattle, and rhinoceros. Together with mouse and human, the segmentation clock periods of the six species did not scale with the animal body weight, but with the embryogenesis length. The biochemical kinetics of the core clock gene HES7 displayed clear scaling with the species-specific segmentation clock period. However, the cellular metabolic rates did not show an evident correlation. Instead, genes involving biochemical reactions showed an expression pattern that scales with the segmentation clock period. Altogether, our stem cell zoo uncovered general scaling laws governing species-specific developmental tempo.

Induction of primordial germ cell-like cells from common marmoset embryonic stem cells by inhibition of WNT and retinoic acid signaling.

Reconstitution of the germ cell lineage using pluripotent stem cells provides a unique platform to deepen our understanding of the mechanisms underlying germ cell development and to produce functional gametes for reproduction. This study aimed to establish a culture system that induces a robust number of primordial germ cell-like cells (PGCLCs) from common marmoset (Callithrix jacchus) embryonic stem cells. The robust induction was achieved by not only activation of the conserved PGC-inducing signals, WNT and BMP4, but also temporal inhibitions of WNT and retinoic acid signals, which prevent mesodermal and neural differentiation, respectively, during PGCLC differentiation. Many of the gene expression and differentiation properties of common marmoset PGCLCs were similar to those of human PGCLCs, making this culture system a reliable and useful primate model. Finally, we identified PDPN and KIT as surface marker proteins by which PGCLCs can be isolated from embryonic stem cells without genetic manipulation. This study will expand the opportunities for research on germ cell development and production of functional gametes to the common marmoset.

Robust induction of primordial germ cells of white rhinoceros on the brink of extinction.

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis.

In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that OVOL2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) that drives gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and, consequently, induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation. This article has an associated 'The people behind the papers' interview.

Direct Observation of Conversion From Walled Cells to Wall-Deficient L-Form and Vice Versa in Escherichia coli Indicates the Essentiality of the Outer Membrane for Proliferation of L-Form Cells.

Gram-negative bacteria such as Escherichia coli are surrounded by an outer membrane, which encloses a peptidoglycan layer. Even if thinner than in many Gram-positive bacteria, the peptidoglycan in E. coli allows cells to withstand turgor pressure in hypotonic medium. In hypertonic medium, E. coli treated with a cell wall synthesis inhibitor such as penicillin G form wall-deficient cells. These so-called L-form cells grow well under anaerobic conditions (i.e., in the absence of oxidative stress), becoming deformed and dividing as L-form. Upon removal of the inhibitor, they return to the walled rod-shaped state. Recently, the outer membrane was reported to provide rigidity to Gram-negative bacteria and to strengthen wall-deficient cells. However, it remains unclear why L-form cells need the outer membrane for growth. Using a microfluidic system, we found that, upon treatment with the outer membrane-disrupting drugs polymyxin B and polymyxin B nonapeptide or with the outer membrane synthesis inhibitor CHIR-090, the cells lysed during cell deformation and division, indicating that the outer membrane was important even in hypertonic medium. L-form cells could return to rod-shaped when trapped in a narrow space, but not in a wide space, likely due to insufficient physical force. Outer membrane rigidity could be compromised by lack of outer membrane proteins; Lpp, OmpA, or Pal. Deletion of lpp caused cells to lyse during cell deformation and cell division. In contrast, ompA and pal mutants could be deformed and return to small oval cells even when less physical force was exerted. These results strongly suggest that wall-deficient E. coli cells require a rigid outer membrane to survive, but not too rigid to prevent them from changing cell shape.

A Conserved Histone H3-H4 Interface Regulates DNA Damage Tolerance and Homologous Recombination during the Recovery from Replication Stress.

In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. In this study, we screened a collection of mutations for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress methyl methanesulfonate (MMS) sensitivity of DDT-deficient cells (referred to here as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with decreased MMS-induced mutagenesis relative to that of wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage.

Cyclin-dependent kinase modulates budding yeast Rad5 stability during cell cycle.

The DNA damage tolerance (DDT) pathway facilitates the bypass of the fork-blocking lesions without removing them through either translesion DNA synthesis or error-free damage bypass mechanism. The Saccharomyces cerevisiae Rad5 is a multi-functional protein involved in the error-free branch of the DDT pathway, and its protein level periodically fluctuates through the cell cycle; however, the mechanistic basis and functional importance of the Rad5 level for the cell cycle regulation remain unclear. Here, we show that Rad5 is predominantly phosphorylated on serine 130 (S130) during S/G2 phase and that this modification depends on the cyclin-dependent kinase Cdc28/CDK1. We also show that the phosphorylated Rad5 species at S130 exhibit a relatively short half-life compared with non-phosphorylated Rad5 moiety, and that the Rad5 protein is partially stabilized in phosphorylation-defective rad5 S130A cells. Importantly, the elimination of this modification results in a defective cell-cycle dependent Rad5 oscillation pattern. Together, our results demonstrate that CDK1 modulates Rad5 stability by phosphorylation during the cell cycle, suggesting a crosstalk between the phosphorylation and degradation of Rad5.

Generation of germ cells from pluripotent stem cells in mammals.

BACKGROUND: The germ cell lineage transmits genetic and epigenetic information to the next generation. Primordial germ cells (PGCs), the early embryonic precursors of sperm or eggs, have been studied extensively. Recently, in vitro models of PGC induction have been established in the mouse. Many attempts are reported to enhance our understanding of PGC development in other mammals, including human. METHODS: Here, original and review articles that have been published on PubMed are reviewed in order to give an overview of the literature that is focused on PGC development, including the specification of in vivo and in vitro in mice, human, porcine, and bovine. RESULTS: Mammalian PGC development, in vivo and in vitro, have been studied primarily by using the mouse model as a template to study PGC specification in other mammals, including human, porcine, and bovine. CONCLUSION: The growing body of published works reveals similarities, as well as differences, in PGC establishment in and between mouse and human.

Role of neuromedin U in accelerating of non-alcoholic steatohepatitis in mice.

Neuromedin U (NMU), a neuropeptide originally isolated from porcine spinal cord, has multiple physiological functions and is involved in obesity and inflammation. Excessive fat accumulation in the liver leads to non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), which is closely associated with obesity. NAFLD and NASH develop and progress via complex pathophysiological processes, and it remains unclear to what extend the NMU system contributes to the risk of obesity-related disorders such as NAFLD and NASH. Here, we demonstrate that the NMU system plays a role in NAFLD/NASH pathogenesis. In the normal mouse liver, NMU mRNA was not detectable, and expression of the mRNA encoding neuromedin U receptor 1 (NMUR1), the peripheral receptor of NMU, was low. However, the expression of both was significantly increased in the livers of NASH mice. Furthermore, overproduction of NMU induced the mouse liver by hydrodynamic injection, exacerbated NASH pathogenesis. These data indicate a novel role for the peripheral NMU system, providing new insights into the pathogenesis of NAFLD/NASH.

Life-threatening emphysematous liver abscess associated with poorly controlled diabetes mellitus: a case report.

BACKGROUND: Emphysematous liver abscesses are defined as liver abscesses accompanied by gas formation. The fatality rate is extremely high at 27%, necessitating prompt intensive care. CASE PRESENTATION: The patient was a 69-year-old Japanese man with type 2 diabetes. He visited the emergency outpatient department for fever and general malaise that had been ongoing for 2 weeks. Abdominal computed tomography revealed an abscess 5 cm in diameter accompanied by gas formation in the right hepatic lobe. Markedly impaired glucose tolerance was observed with a blood sugar level of 571 mg/dL and a glycated hemoglobin level of 14.6%. The patient underwent emergency percutaneous abscess drainage, and intensive care was subsequently initiated. Klebsiella pneumoniae was detected in both the abscess cavity and blood cultures. The drain was removed 3 weeks later, and the patient was discharged. CONCLUSION: Emphysematous liver abscesses are often observed in patients with poorly controlled diabetes, and the fatality rate is extremely high. Fever and malaise occasionally mask life-threatening infections in diabetic patients, necessitating careful examination.

Electrocapillary Phenomena at Edible Oil/Saline Interfaces.

Interfacial tension between edible oil and saline was measured under applied electric fields to understand the electrocapillary phenomena at the edible oil/saline interfaces. The electric responses of saline droplets in edible oil were also observed microscopically to examine the relationship between the electrocapillary phenomena and interfacial polarization. When sodium oleate (SO) was added to edible oil (SO-oil), the interfacial tension between SO-oil and saline decreased. However, no decrease was observed for additive-free oil or oleic acid (OA)-added oil (OA-oil). Microscopic observations suggested that the magnitude of interfacial polarization increased in the order of additive-free oil < OA-oil < SO-oil. The difference in electrocapillary phenomena between OA- and SO-oils was closely related to the polarization magnitude. In the case of SO-oil, the decrease in interfacial tension was remarkably larger for saline (pH 5.4~5.6) than that for phosphate-buffered saline (PBS, pH 7.2~7.4). However, no difference was observed between the electric responses of PBS and saline droplets in SO-oil. The difference in electrocapillary phenomena for PBS and saline could not be simply explained in terms of polarization magnitude. The ratio of ionized and non-ionized OA at the interfaces changed with the saline pH, possibly leading to the above difference.

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3/4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRß, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

Successful conservative treatment of emphysematous gastritis.

Emphysematous gastritis is an extremely rare disease with an unfavorable prognosis. To date, very few studies have been conducted regarding the intragastric recovery process based on endoscopic findings. We herein report a case of emphysematous gastritis that improved with long-term (five months) conservative treatment in which we were able to observe the intragastric recovery process endoscopically. In cases in which emphysematous gastritis is suspected, it is important to provide prompt diagnostic imaging (including CT) and early appropriate treatment in order to improve the prognosis.

Oxidation of aromatic N-heterocyclic compounds to N-oxides by Verticillium sp. GF39 cells.

Verticillium sp. GF39, catalyzing the oxidation of 1-methylisoquinoline to 1-methylisoquinoline N-oxide, was found to be the highest N-oxide producer. Under the optimized reaction conditions, the whole cells of Verticillium sp. GF39 formed 5 mM 1-methylisoquinoline N-oxide from 1-methylisoquinoline with a molar conversion yield of 100% after a 10-h incubation at 20°C. The whole cells also acted on pyridine, 2-methylpyridine, quinoline and isoquinoline and formed the corresponding N-oxides.