C57BL/6 mouse is the most standard strain in mouse genetics. The strain does, however, have several disadvantages; one being the difficulty in establishing embryonic stem (ES) cells. No reliable C57BL/6 ES cell line is widely available for creating mutant mice through gene targeting. It also greatly favors mouse genetics if one can routinely make multiple mutations by stably culturing germline-competent C57BL/6 ES cells or if one can routinely establish ES cells from C57BL/6-derived mutant mice to make multiple mutations. Recently, an ES culture method with three inhibitors (3i: SU5402 for FGFR, PD184352 for ERK, and CHIR99021 for GSK3) has been reported. Here we show that this 3i method is extremely instrumental in establishing and culturing germline-competent ES cells in the C57BL/6N strain.

The proliferation of intestinal stem cells is maintained by EGF, a key component of the medium required to support organoids ex vivo. Here, we identified that NRG1, not EGF, is up-regulated during tissue repair following injury. NRG1 is expressed in mesenchymal stromal cells, macrophages and Paneth cells. Knockout of NRG1 produced a decrease in cell proliferation within crypts and a reduced capacity to regenerate following injury. Treatment with NRG1 robustly stimulated proliferation in crypts and budding of organoids. Molecular characterisation of the effects of NRG1 revealed an elevated and sustained activation of MAPK and AKT and a proliferative signature. NRG1 also had a strong impact on the expression of stem cell markers, the ability of progenitor cells to initiate organoid growth and enhanced regeneration. Our data suggest mesenchymal-derived NRG1 is a potent mediator of tissue regeneration and may inform the development of NRG1-based therapies for enhancing intestinal repair after injury.

(10,11,27,28).We have shown that the switch from pigmented retinal cells of 9-day-old check embryos into lens cells occurs in clonal cell culture without interactions with other cell types (14). The question then arises whether this approach can be extended to iris epithelial cells of newts which possess the capacity for Wolffian regeneration in vivo. This communication describes the differentiation of lens-like structures in cultures of dissociated iris epithelial cells of adult newts. MATERIALS AND METHODSTwenty-five to 50 adult newts, Cynops (Triturus) pyrrhogaster were used in each experiment. Isolated whole eyes were sterilized by three 1-min immersions in 70% ethanol. The irisrings (iris pars iridica) were then removed (11) and treated in an enzyme mixture that contained 5 parts of 0.4% (v/v) Diluted Leibovitz medium IL15 (12) was used as the culture medium and was prepared by dissolving 0.894 g of commercial Leibovitz L-15 powder (GIBCO) in 90 ml of triple-distilled water; this was supplemented with 10 ml of fetal calf serum (GIBCO, Control No. A322102), 3200 IU of penicillin, and 4 mg of streptomycin before filtration by Millipore filter.For the immunological identification of lens products in cultured cells, antisera were prepared in rabbits against the 15,000 X g supernatant of adult newt lenses in 20 mM Tris HCl buffer (pH 7.5) and against the a-, f-, and y-crystallin fractions, respectively. The separation of crystallins from the whole lens extract was made by column chromatography through Sephadex G-50 and G-200, and DEAE-Sephadex A-50 according to established methods (13). Sixteen rabbits were injected, one each with 40, 5, 3, and 1 mg of each protein fraction, respectively. Each rabbit received injections of protein with an incomplete Freund's adjuvant at intervals of two weeks. Antisera were usually obtained 10 days-after the second injection. In immunodiffusion tests (15) anti-whole lens extract and anti-a-crystallin cross-reacted faintly with extracts of heterologous organs of newts. This cross-reactivity was removed by absorbing the antisera with saline extracts of liver homogenate of adult newts. Cross-reactions of anti-acrystallin with the heterologous crystallins were also removed by absorption with 0-crystallin solution, whereas anti-acrystallin and anti-y-crystallin did not cross-react with the heterologous test-antigens (Fig. 1)

The aim of the present study was to reveal the spatiotemporal relations among cortical regions involved in the initiation of voluntary swallowing in humans using magnetoencephalography (MEG). As a control task, finger extension movement, which is purely voluntary, was also investigated using the same techniques. The swallowing-related activity was distributed widely for 2000 ms before the electromyogram onset of the right suprahyoid muscle; however, the finger-related activity occurred in the late stage of the recording. The cingulate cortex, the insula, and the inferior frontal gyrus were the main loci active prior to swallowing. These cortical loci coincide with those suggested by previous human brain mapping studies that investigated the brain mechanism which controls swallowing. Activation in the cingulate cortex was registered in the early stage of swallowing and could be related to the cognitive process regarding the food being safe to swallow. The activation in the insula lasted for a long time before the initiation of swallowing. This suggests that the long-lasting activation in the insula prior to swallowing is essential for the initiation of swallowing.

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