Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

[Novel Application of Post-contrast T1map for Detection of Subendocardial Infarction].

In cardiac magnetic resonance (CMR) for myocardial infarction, there have been quite a few cases of obscure image contrast between subendocardial lesion and left ventricular (LV) blood pool on late gadolinium enhancement (LGE) images. This study was motivated by confirmation of usefulness of post-contrast T1map for detection of subendocardial infarction. From June 2017 to May 2018, forty-eight consecutive patients who underwent contrast-enhanced CMR to assess myocardial infarction were reviewed. We measured the contrast ratio (CR) between the infarcted myocardium and LV blood pool on LGE and on post-contrast T1map images, and compared them. The CR (mean±standard deviation) was -0.04±0.11 for LGE images and 0.02±0.04 for post-contrast T1map images (P<0.05). These results suggest that the post-contrast T1map, which uses the difference in T1 value as image contrast rather than magnitude image, can clearly depict the boundary between the infarcted myocardium and LV blood pool. The addition of post-contrast T1map to image interpretation might provide valuable information in the evaluation of subendocardial infarction.

Exploring the human extraembryonic mesoderm using naive pluripotent stem cells.

In this issue of Cell Stem Cell, Pham et al. report that naive human pluripotent stem cells can be differentiated into extraembryonic mesoderm cells (EXMCs). EXMCs are maintained for up to 70 days, resemble human and monkey extraembryonic mesoderm, and provide a model to study EXMC specification and function.

Valproic acid up-regulates the whole NO-citrulline cycle for potent iNOS-NO signaling to promote neuronal differentiation of adipose tissue-derived stem cells.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.

Clinical significance of gadobutrol in magnetic resonance imaging for the detection of myocardial infarction: matched-pair cohort study to compare with gadopentetate dimeglumine at standard dose.

We aimed to confirm whether gadobutrol is more useful for late gadolinium enhancement (LGE) imaging than gadopentetate dimeglumine (Gd-DTPA) at the standard dose. Patients who underwent LGE imaging to assess myocardial infarction were retrospectively enrolled: gadobutrol, 51 cases; Gd-DTPA, 49 cases. Contrast ratios of infarcted lesion to remote myocardium (CRremote) and to left ventricular blood (CRblood) were compared. Patient characteristics that might affect image contrast did not differ between groups. CRremote (median, (interquartile range)) at 10 and 15 min after administration was 0.79 (0.08) and 0.70 (0.09) for gadobutrol, and 0.74 (0.13) and 0.65 (0.16) for Gd-DTPA (P < 0.05 and < 0.05), respectively. CRblood was - 0.05 (0.17) and - 0.002 (0.15) for gadobutrol, and - 0.05 (0.18) and 0.01 (0.16) for Gd-DTPA (P = 0.29 and = 0.22), respectively. Gadobutrol provided significantly better delineation of infarcted from normal myocardium than Gd-DTPA. Meanwhile, there was no difference in image contrast between infarcted myocardium and left ventricular blood.

Valproic acid promotes mature neuronal differentiation of adipose tissue-derived stem cells through iNOS-NO-sGC signaling pathway.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing ßIII-tubulin protein, a neuronal marker, by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2 h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400 W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as ßIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS-NO-sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.

Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone deacetylase inhibitor.

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker ßIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.