Journal: Scientific Reports

Article Title: Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

doi: 10.1038/s41598-019-52204-7

Figure Lengend Snippet: Characterization of vascularized neural organoids. Neural spheres consisting of Sox1 + neuroepithelial cells ( A ) and Brachyury + mesodermal spheres. ( B ) Spheres were brought in co-culture. The formation of chimeric neuro-mesodermal aggregates was observed ( C ). The aggregates were cultured for up to 280 days ( D ). ( E ) HE-staining of sections showing the interface between neuroepithelial (left side) and mesenchymal part (right side). ( F – G ) Immunofluorescence analyses showing the Pax6 + neuroepithelium and CD31 + endothelial cells. ( H – D ) The neural part consists of Sox1 + stem cells and TUJ1 + neurons. ( J – K ) At the interface between neuroepithelium and mesenchyme, CD31 + vessels form a perineural plexus. ( L ) A capillary at the perineural plexus in an ED 5 chicken embryo is depicted. Blood cells show green autofluorescence. ( M ) Within the mesodermal part an extensive network of blood vessels can be observed. M shows a maximum intensity projection of a whole mount stained organoid. ( N ) HE staining of paraffin sections from the mesodermal part of an organoid. The picture shows a vessel structure with clear lumen (asterisk) and a small capillary (arrowhead). ( O – S ) Transmission electron microscopic pictures showing the endothelium of vessel structures within the organoids. *Periendothelial cells, EC: endothelial cells, BM: basement membrane, IJ: intercellular junction, CV: caveolae, MV: microvesicle.

Article Snippet: Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used.

Techniques: Co-Culture Assay, Cell Culture, Staining, Immunofluorescence, Transmission Assay, Membrane

Journal: Scientific Reports

Article Title: Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

doi: 10.1038/s41598-019-52204-7

Figure Lengend Snippet: Characterization of the neural part of the organoid. Pictures show organoids at culture day 210. ( A ) HE-stained sections reveal ventricle-like cavities inside the neural tissue. ( B – C ) Neuroepithelial cells lining the ventricle-like structures stain positive for Sox1 and Nestin, while the cells surrounding the stem cell zone express the neuronal marker protein MAP2. ( C ) Shows a higher magnification of B. ( D ) The apical side of the neuroepithelium is marked by N-Cadherin + cell-cell junctions. TUJ1 + cells can be found towards the basal side indicating neuronal differentiation. ( E ) Besides TUJ1 + neurons, GFAP + radial glia cells or astrocytes can be found located towards the basal side of the epithelium.

Article Snippet: Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used.

Techniques: Staining, Marker

Journal: Scientific Reports

Article Title: Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

doi: 10.1038/s41598-019-52204-7

Figure Lengend Snippet: Vascularization of the neural part of the organoid and invasion of microglia-like cells. ( A ) At day 180 a CD31 + endothelial network infiltrating the Pax6 + neural part of the organoid was observed. ( B ) shows a higher magnification of A. ( C ) Infiltrating vessels were found within the neural tissue surrounding Sox1 + neuroepithelial structures. ( D – E ) CD31 + capillaries within the neural part of the organoid closely associated with GFAP + astrocytes/radial glia and TUJ1 + neurons. ( F ) GFAP + cells form close contacts with CD31 + endothelial cells (see inset). ( G ) Iba1 + macrophage-like cells were observed in close association with CD31 + capillaries within the mesodermal part of the organoid. ( H ) Iba1 + cells were also found within the neural part of the organoid being tightly associated with TUJ1 + neurons suggesting a microglia-like identity. ( I ) Microglia-like cells show a cellular morphology similar to microglial cells at the transition from an amoeboid to a ramified appearance. ( A , B , F , G , H ) Show maximum intensity projections of whole-mount stained cleared organoids. ( C , D , E , I) show stained paraffin sections.

Article Snippet: Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used.

Techniques: Staining

Journal: Yonsei Medical Journal

Article Title: Changes in Inward Rectifier K + Channels in Hepatic Stellate Cells During Primary Culture

doi: 10.3349/ymj.2008.49.3.459

Figure Lengend Snippet: Relative inward rectifier K + channel and SUR gene expression in HSC. (A) The inward rectifier K + channel α-subunit gene expression in HSC was measured using real-time RT-PCR. The bar graphs show the relative gene expression for each inward rectifier K + channel subfamily (K ir 1.1, K ir 2.1 - K ir 2.4, K ir 3.1 - K ir 3.4, K ir 4.1 - K ir 4.2, K ir 5.1, K ir 6.1 - K ir 6.2, and K ir 7.1). (B) SUR gene expression was measured. The bar graphs show the relative gene expression for SUR1, SUR2A, and SUR2B. The HSC were used at 1 day, 1 week, 2 weeks, and 3 weeks of culture. The expression levels were normalized to GAPDH and calibrated by K ir 2.1 expression in the HSC cultured for 1 day. The data are shown as the mean ± SEM (n = 3). (C) K ir 2.1 and K ir 6.1 protein expression were measured by Western blotting. There was a band (55 kD) in the anti-K ir 2.1 membrane (Upper) as well as the anti-K ir 6.1 membrane (Lower). Both membranes were re-probed with anti-GAPDH antibody as shown. The data is representative of three independent experiments.

Article Snippet: The anti-K ir 2.1 and anti-K ir 6.1 antibodies were obtained from Alomone Labs (Jerusalem, Israel), the anti-GAPDH antibody was acquired from Novus Biologicals (Littleton, CO, USA), and Western Lightning was supplied by PerkinElmer Life Sciences (Gaithersburg, MD, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Membrane