Reconstruction and deconstruction of human somitogenesis in vitro.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.

Temperature-dependent sex determination is mediated by pSTAT3 repression of Kdm6b.

In many reptiles, including the red-eared slider turtle Trachemys scripta elegans (T. scripta), sex is determined by ambient temperature during embryogenesis. We previously showed that the epigenetic regulator Kdm6b is elevated at the male-producing temperature and essential to activate the male pathway. In this work, we established a causal link between temperature and transcriptional regulation of Kdm6b We show that signal transducer and activator of transcription 3 (STAT3) is phosphorylated at the warmer, female-producing temperature, binds the Kdm6b locus, and represses Kdm6b transcription, blocking the male pathway. Influx of Ca2+, a mediator of STAT3 phosphorylation, is elevated at the female temperature and acts as a temperature-sensitive regulator of STAT3 activation.

Generation of reactive oxygen species contributes to the development of carbon black cytotoxicity to vascular cells.

Carbon black, a particulate form of pure elemental carbon, is an industrial chemical with the high potential of occupational exposure. Although the relationship between exposure to particulate matters (PM) and cardiovascular diseases is well established, the cardiovascular risk of carbon black has not been characterized clearly. In this study, the cytotoxicity of carbon black to vascular smooth muscle and endothelial cells were examined to investigate the potential vascular toxicity of carbon black. Carbon black with distinct particle size, N330 (primary size, 28~36 nm) and N990 (250~350 nm) were treated to A-10, rat aortic smooth muscle cells and human umbilical vein endothelial cell line, ECV304, and cell viability was assessed by lactate dehydrogenase (LDH) leakage assay. Treatment of carbon black N990 resulted in the significant reduction of viability in A-10 cells at 100 µg/ml, the highest concentration tested, while N330 failed to cause cell death. Cytotoxicity to ECV304 cells was induced only by N330 at higher concentration, 200 µg/ml, suggesting that ECV304 cells were relatively resistant to carbon black. Treatment of 100 µg/ml N990 led to the elevation of reactive oxygen species (ROS) detected by dichlorodihydrofluorescein (DCF) in A-10 cells. Pretreatment of antioxidants, N-acetylcysteine (NAC) and sulforaphane restored decreased viability of N990-treated A-10 cells, and N-acetylcysteine, but not sulforaphane, attenuated N990-induced ROS generation in A-10 cells. Taken together, present study shows that carbon black is cytotoxic to vascular cells, and the generation of reactive oxygen contributes to the development of cytotoxicity. ROS scavenging antioxidant could be a potential strategy to attenuate the toxicity induced by carbon black exposure.