Reconstituting human somitogenesis in vitro.

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms; however, the details of this process in humans remain largely unknown owing to ethical and technical limitations. Despite recent advances with pluripotent stem cell-based approaches1-5, models that robustly recapitulate human somitogenesis in both space and time remain scarce. Here we introduce a pluripotent stem cell-derived mesoderm-based 3D model of human segmentation and somitogenesis-which we termed 'axioloid'-that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF-WNT signalling gradients and retinoic acid signalling components. We identify an unexpected critical role of retinoic acid signalling in the stabilization of forming segments, indicating distinct, but also synergistic effects of retinoic acid and extracellular matrix on the formation and epithelialization of somites. Comparative analysis demonstrates marked similarities of axioloids to the human embryo, further validated by the presence of a Hox code in axioloids. Finally, we demonstrate the utility of axioloids for studying the pathogenesis of human congenital spine diseases using induced pluripotent stem cells with mutations in HES7 and MESP2. Our results indicate that axioloids represent a promising platform for the study of axial development and disease in humans.

Cloud service checklist for academic communities and customization for genome medical research.

In this paper, we present a cloud service checklist designed to help IT administrators or researchers in academic organizations select the most suitable cloud services. This checklist, which comprises items that we believe IT administrators or researchers in academic organizations should consider when they adopt cloud services, comprehensively covers the issues related to a variety of cloud services, including security, functionality, performance, and law. In response to the increasing demands for storage and computing resources in genome medical science communities, various guidelines for using resources operated by external organizations, such as cloud services, have been published by different academic funding agencies and the Japanese government. However, it is sometimes difficult to identify the checklist items that satisfy the genome medical science community's guidelines, and some of these requirements are not included in the existing checklists. This issue provided our motivation for creating a cloud service checklist customized for genome medical research communities. The resulting customized checklist is designed to help researchers easily find information about the cloud services that satisfy the guidelines in genome medical science communities. Additionally, we explore whether many cloud service providers satisfy the requirements or checklist items in the cloud service checklist for genome medical research by evaluating their survey responses.

Club Cells Are the Primary Target for Permethrin-Induced Mouse Lung Tumor Formation.

Permethrin has been shown to increase lung adenomas in female CD-1 mice, but not in male mice or Wistar rats. The proposed mode of action (MOA) for permethrin-induced female mouse lung tumor formation involves morphological changes in Club cells; increased Club cell proliferation; increased Club cell hyperplasia, and lung tumor formation. In this study, the treatment of female CD-1 mice with tumorigenic doses (2500 and 5000 ppm) of permethrin, but not with a nontumorigenic dose (20 ppm), for 14 and/or 28 days increased Club cell replicative DNA synthesis. Global gene expression analysis of female mouse lung samples demonstrated that permethrin treatment up-regulated 3 genes associated with cell proliferation, namely aldehyde dehydrogenase 3a1 (Aldh3a1), oxidative stress-induced growth inhibitor 1, and thioredoxin reductase 1. Treatment with 2500 and 5000 ppm, but not 20 ppm, permethrin for 7 days produced significant increases in mRNA levels of these 3 genes. Immunohistochemical analysis demonstrated that Club cell secretory protein, CYP2F2, and ALDH3A1 colocalized in Club cells; confirmed by flow cytometry analysis of lung cells employing KI67 as a cell proliferation marker. Overall, the present data extend the proposed MOA by demonstrating that Club cells are the primary initial target of permethrin administration in female mouse lungs. As humans are quantitatively much less sensitive to agents that increase Club cell proliferation and lung tumor formation in mice, it is most likely that permethrin could not produce lung tumors in humans. This conclusion is supported by available negative epidemiological data from several studies.

Imaging and manipulating the segmentation clock.

During embryogenesis, the processes that control how cells differentiate and interact to form particular tissues and organs with precise timing and shape are of fundamental importance. One prominent example of such processes is vertebrate somitogenesis, which is governed by a molecular oscillator called the segmentation clock. The segmentation clock system is initiated in the presomitic mesoderm in which a set of genes and signaling pathways exhibit coordinated spatiotemporal dynamics to establish regularly spaced boundaries along the body axis; these boundaries provide a blueprint for the development of segment-like structures such as spines and skeletal muscles. The highly complex and dynamic nature of this in vivo event and the design principles and their regulation in both normal and abnormal embryogenesis are not fully understood. Recently, live-imaging has been used to quantitatively analyze the dynamics of selected components of the circuit, particularly in combination with well-designed experiments to perturb the system. Here, we review recent progress from studies using live imaging and manipulation, including attempts to recapitulate the segmentation clock in vitro. In combination with mathematical modeling, these techniques have become essential for disclosing novel aspects of the clock.

Species-specific segmentation clock periods are due to differential biochemical reaction speeds.

Although mechanisms of embryonic development are similar between mice and humans, the time scale is generally slower in humans. To investigate these interspecies differences in development, we recapitulate murine and human segmentation clocks that display 2- to 3-hour and 5- to 6-hour oscillation periods, respectively. Our interspecies genome-swapping analyses indicate that the period difference is not due to sequence differences in the HES7 locus, the core gene of the segmentation clock. Instead, we demonstrate that multiple biochemical reactions of HES7, including the degradation and expression delays, are slower in human cells than they are in mouse cells. With the measured biochemical parameters, our mathematical model accounts for the two- to threefold period difference between the species. We propose that cell-autonomous differences in biochemical reaction speeds underlie temporal differences in development between species.

Publisher Correction: Methylation deficiency disrupts biological rhythms from bacteria to humans.

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

Methylation deficiency disrupts biological rhythms from bacteria to humans.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.

Coupling delay controls synchronized oscillation in the segmentation clock.

Individual cellular activities fluctuate but are constantly coordinated at the population level via cell-cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM)1,2. This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion3-7. However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe (Lfng) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng-null PSM. However, when Lfng-null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell-cell coupling. Mixed cultures of control and Lfng-null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results-together with mathematical modelling-raised the possibility that Lfng-null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators8. Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng-null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation.

In vitro characterization of the human segmentation clock.

The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock1,2. Although this oscillator has been well-characterized in model organisms1,2, whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation defects have implicated several human orthologues of cyclic genes that are associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans3. Here we show that human PSM cells derived in vitro-as well as those of the mouse4-recapitulate the oscillations of the segmentation clock. Human PSM cells oscillate with a period two times longer than that of mouse cells (5 h versus 2.5 h), but are similarly regulated by FGF, WNT, Notch and YAP signalling5. Single-cell RNA sequencing reveals that mouse and human PSM cells in vitro follow a developmental trajectory similar to that of mouse PSM in vivo. Furthermore, we demonstrate that FGF signalling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in 'clock and wavefront' models1. Our work identifying the human segmentation clock represents an important milestone in understanding human developmental biology.

Dynamic Delta-like1 expression in presomitic mesoderm cells during somite segmentation.

During somite segmentation, the expression of clock genes such as Hes7 oscillates synchronously in the presomitic mesoderm (PSM). This synchronous oscillation slows down in the anterior PSM, leading to wave-like propagating patterns from the posterior to anterior PSM. Such dynamic expression depends on Notch signaling and is critical for somite formation. However, it remains to be determined how slowing oscillations in the anterior PSM are controlled, and whether the expression of the Notch ligand Delta-like1 (Dll1) oscillates on the surface of individual PSM cells, as postulated to be responsible for synchronous oscillation. Here, by using Dll1 fluorescent reporter mice, we performed live-imaging of Dll1 expression in PSM cells and found the oscillatory expression of Dll1 protein on the cell surface regions. Furthermore, a comparison of live-imaging of Dll1 and Hes7 oscillations revealed that the delay from Dll1 peaks to Hes7 peaks increased in the anterior PSM, suggesting that the Hes7 response to Dll1 becomes slower in the anterior PSM. These results raise the possibility that Dll1 protein oscillations on the cell surface regulate synchronous Hes7 oscillations, and that the slower response of Hes7 to Dll1 leads to slower oscillations in the anterior PSM.

Small-molecule screening yields a compound that inhibits the cancer-associated transcription factor Hes1 via the PHB2 chaperone.

The transcription factor Hes family basic helix-loop-helix transcription factor 1 (Hes1) is a downstream effector of Notch signaling and plays a crucial role in orchestrating developmental processes during the embryonic stage. However, its aberrant signaling in adulthood is linked to the pathogenesis of cancer. In the present study, we report the discovery of small organic molecules (JI051 and JI130) that impair the ability of Hes1 to repress transcription. Hes1 interacts with the transcriptional corepressor transducing-like enhancer of split 1 (TLE1) via an interaction domain comprising two tryptophan residues, prompting us to search a chemical library of 1,800 small molecules enriched for indole-like π-electron-rich pharmacophores for a compound that blocks Hes1-mediated transcriptional repression. This screening identified a lead compound whose extensive chemical modification to improve potency yielded JI051, which inhibited HEK293 cell proliferation with an EC50 of 0.3 µm Unexpectedly, using immunomagnetic isolation and nanoscale LC-MS/MS, we found that JI051 does not bind TLE1 but instead interacts with prohibitin 2 (PHB2), a cancer-associated protein chaperone. We also found that JI051 stabilizes PHB2's interaction with Hes1 outside the nucleus, inducing G2/M cell-cycle arrest. Of note, JI051 dose-dependently reduced cell growth of the human pancreatic cancer cell line MIA PaCa-2, and JI130 treatment significantly reduced tumor volume in a murine pancreatic tumor xenograft model. These results suggest a previously unrecognized role for PHB2 in the regulation of Hes1 and may inform potential strategies for managing pancreatic cancer.

ES cell-derived presomitic mesoderm-like tissues for analysis of synchronized oscillations in the segmentation clock.

Somites are periodically formed by segmentation of the anterior parts of the presomitic mesoderm (PSM). In the mouse embryo, this periodicity is controlled by the segmentation clock gene Hes7, which exhibits wave-like oscillatory expression in the PSM. Despite intensive studies, the exact mechanism of such synchronous oscillatory dynamics of Hes7 expression still remains to be analyzed. Detailed analysis of the segmentation clock has been hampered because it requires the use of live embryos, and establishment of an in vitro culture system would facilitate such analyses. Here, we established a simple and efficient method to generate mouse ES cell-derived PSM-like tissues, in which Hes7 expression oscillates like traveling waves. In these tissues, Hes7 oscillation is synchronized between neighboring cells, and the posterior-anterior axis is self-organized as the central-peripheral axis. This method is applicable to chemical-library screening and will facilitate the analysis of the molecular nature of the segmentation clock.

[Development of alternatives to animal experiments using pluripotent stem cells].

Animal experiments have occupied an important position in the safety assessment of chemicals. However, due to the rise in animal welfare as seen in the ban of animal experiments in European cosmetic development, the development of alternative methods for animal experiments has become very important in recent years. Development of in vitro tests for local toxicity such as irritation and sensitization tests is preceded. Meanwhile, alternative tests for systemic toxicity such as chronic and developmental toxicities are under development. In developing alternative methods using cultured cells, we have been focusing on pluripotent stem cells such as ES and iPS cells and studying alternatives to developmental toxicity and neurotoxicity. As an alternative test of developmental toxicity, we developed the Hand 1-Luc EST, which is a simple test utilizing cardiomyocyte differentiation process of mouse ES cells, and Tubb 3- and Reln-Luc ESTs using nerve differentiation process. Recently, it was clarified that the combination of the Hand 1-Luc EST and the Tubb 3- and Reln-Luc ESTs improves the prediction of the developmental toxicity. In the study of in vitro neurotoxicity test using neurons derived from mouse ES cells, evaluation methods for neurite outgrowth using high-content imaging technology and for neural function using multi-electrode arrays were developed. In addition, we introduce differentiation methods for retinal tissues from human ES/iPS cells, which are the results as the collaboration with RIKEN and the present state of an in vitro phototoxicity test using retinal pigment epithelial cells (RPE) derived from human ES cells.

Editor's Highlight: Development of Novel Neural Embryonic Stem CellTests for High-Throughput Screening of Embryotoxic Chemicals.

There is a great demand for appropriate alternative methods to rapidly evaluate the developmental and reproductive toxicity of a wide variety of chemicals. We used the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes as a basis for establishing a rapid and highly reproducible invitro embryotoxicity test known as the Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST). In this study, we developed novel neural-Luc ESTs using two marker genes for neural development, tubulin beta-3 (Tubb3) and Reelin (Reln), and evaluated the capacity of these tests to predict developmental toxicity. In addition, we tested whether an integrated approach (a combination of neural-Luc ESTs and the Hand1-Luc EST) improved developmental toxicant detection. To perform our neural-Luc ESTs, we needed to generate stable transgenic mESCs with individual promoters linked to the luciferase gene, and to establish that similar changes in promoter activities and mRNA expression levels occur during neural differentiation. Based on the concentration-response curves of 15 developmental toxicants and 17 non-developmental toxic chemicals, we derived a prediction formula and assessed the capacity of this formula to predict developmental toxicity. Although both were highly sensitive and specific for predicting developmental toxicity, neural-Luc ESTs had similar predictive capacities. In contrast, neural-Luc ESTs and Hand1-Luc EST had significantly different predictive powers. As expected, the combination of these ESTs increased the sensitivity of developmental toxicant detection. These results demonstrate the convenience and the usefulness of this combination of ESTs as an alternative assay system for future toxicological and mechanistic studies of developmental toxicity.

A novel marker for undifferentiated human embryonic stem cells.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs. The antigen recognized by MAb2 is expressed on the cell surface of undifferentiated hESCs; three diffused bands with molecular mass between 30 and 60 kDa in the lysates of hESCs were diminished during hESC differentiation into neural cells. The expression of MAb2 antigen was also observed on the plasma membrane of lung cancer cells, and MAb2 detected 55, 50, and 35 kDa protein bands in the cell lysates. Immunoprecipitation followed by proteomics analyses identified CD147/basigin as a MAb2 antigen. Finally, the positive expression of CD147/basigin protein in undifferentiated hESCs was confirmed. These results suggested that CD147/basigin could be another undifferentiated hESC marker.

Role of Etv2-positive cells in the remodeling morphogenesis during vascular development.

Etv2 is a critical determinant for the commitment of endothelial (EC) and hematopoietic (HPC) cells from mesoderm. Etv2 is assumed to be transiently required for EC commitment but dispensable after most ECs differentiate around E9.5. To confirm the time window of Etv2 requirement, Etv2 was ablated at different time points using ROSA26CreER mice. Unexpectedly, Etv2 ablation at E9.5 caused vascular remodeling defects in cranial and yolk sac vasculature. Immunostaining showed that Etv2+/VE-cadherin (VECAD)- cells were present around forming vasculature, mostly co-expressing Flk-1 with a small number of Etv2+/VECAD+ cells, indicating that Etv2+/Flk-1+/VECAD- cells are the major Etv2+ population promoting vascular remodeling around E9.5. Gene expression analysis showed up-regulation of Fgf proteins, Il-6, Glypican-3 and matrix metalloproteases in Etv2+/VEDAC- cells over Etv2-/VECAD+ mature ECs. Blockade of those factors caused reduced EC sprouting in ex vivo explant culture from E9.5 embryos, suggesting the functional significance of environmental factors derived from Etv2+ cells. Altogether, we propose that Etv2+/VEDAC- cells around E9.5-E10.5 provide extracellular factors to complete vascular morphogenesis in addition to becoming differentiated ECs incorporated into vessels. This insight for the new role of Ets protein in perivascular Flk-1+/VECAD-/(Etv2+) cells to induce expression of angiogenic factors may provide another strategy to control angiogenesis.

Region-specific Etv2 ablation revealed the critical origin of hemogenic capacity from Hox6-positive caudal-lateral primitive mesoderm.

Hematopoietic cells (HPCs) develop from hemogenic endothelial cells (ECs), a specialized type of ECs undergoing hematopoietic transition. However, the mesoderm origin for hemogenic ECs or HPCs has not been clarified. To examine the origin for hemogenic mesoderm, we inactivated Etv2, a master regulator for EC/HPC commitment, in specific regions. Region-specific Etv2 ablation in early mesoderm caused local EC differentiation block, resulting in the loss of specific vascular beds without compensatory migration of residual ECs into avascular area. This feature of local EC/HPC differentiation block was correlated to the hemogenic potential of each mesoderm subset. We found that caudal-lateral mesoderm of E7.5-8.5 embryos represent the pre-committed population critical for generating hemogenic ECs. Etv2 ablation in caudal-lateral mesoderm by Hoxb6 Cre or Hoxb6CreER transgene affected vitelline plexus formation and intra-aortic hematopoietic clusters. In differentiated embryonic stem cells, this mesoderm subset marked by Hoxb6-lateral mesoderm promoter showed enriched T lymphopoietic potential among Flk-1(+) cells, which could be regarded as a characteristic for definitive HPCs. These findings indicate that critical mesoderm precursors possibly for definitive type hemogenic ECs are regionally specified in primitive mesoderm, suggesting that Hoxb6(+) caudal-lateral mesoderm represents the critical source of HPCs, which are potentially useful to enrich definitive HPCs from embryonic stem cells.

Tracheoplasty using a metallic fusiform centrally-doubled coiled stent for a major tracheal defect in rabbits.

BACKGROUND/PURPOSE: The long-term survival rate of rabbits treated with a fusiform metallic coil for a large tracheal wall defect is 60%. In the present study, the central half of a simple coil was covered by a double coil to prevent the ingrowth of the surrounding connective tissue and to provide a sutureless fixation of the coil to obtain a further increase in the survival rate. STUDY DESIGN: The anterior half of the tracheal wall was removed for a longitudinal length of 6 tracheal rings to make a large tracheal defect. Metallic coils were placed into the tracheal lumen through the wall defect. The doubly-coiled portion was designed to fit the length of the defect to eliminate the need for suture fixation. The survival at two months after the operation, endoscopic findings and histological changes were evaluated. RESULTS: All 6 rabbits treated with a centrally-doubled coiled stent survived without major respiratory trouble for at least 2 months. Endoscopic examinations performed 1 month after the operation revealed an irregular coating of reddish granulation tissue inside the coil, and a wet portion was aspirated. The tracheal defect was replaced with fibrous tissue, but neither a complete epithelialization nor cartilage formation was observed. CONCLUSION: These results suggest that the metallic coil was useful to obtain an improvement in the survival of rabbits with a large tracheal wall defect. Therefore, this new coil might be indicated for the treatment of tracheal wall defects.

Relationship between subjective fall risk assessment and falls and fall-related fractures in frail elderly people.

BACKGROUND: Objective measurements can be used to identify people with risks of falls, but many frail elderly adults cannot complete physical performance tests. The study examined the relationship between a subjective risk rating of specific tasks (SRRST) to screen for fall risks and falls and fall-related fractures in frail elderly people. METHODS: The SRRST was investigated in 5,062 individuals aged 65 years or older who were utilized day-care services. The SRRST comprised 7 dichotomous questions to screen for fall risks during movements and behaviours such as walking, transferring, and wandering. The history of falls and fall-related fractures during the previous year was reported by participants or determined from an interview with the participant's family and care staff. RESULTS: All SRRST items showed significant differences between the participants with and without falls and fall-related fractures. In multiple logistic regression analysis adjusted for age, sex, diseases, and behavioural variables, the SRRST score was independently associated with history of falls and fractures. Odds ratios for those in the high-risk SRRST group (≥ 5 points) compared with the no risk SRRST group (0 point) were 6.15 (p < 0.01) for a single fall, 15.04 (p < 0.01) for recurrent falls, and 5.05 (p < 0.01) for fall-related fractures. The results remained essentially unchanged in subgroup analysis accounting for locomotion status. CONCLUSION: These results suggest that subjective ratings by care staff can be utilized to determine the risks of falls and fall-related fractures in the frail elderly, however, these preliminary results require confirmation in further prospective research.