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. 2016 Mar 8;7:10774. doi: 10.1038/ncomms10774

Figure 4. ISL1+ CVP differentiates and forms human–mouse chimeric circulatory system in vivo.

Figure 4

(a) Xenografts stained positive for GFP present in kidney capsule and adult mouse heart after myocardial infarction (MI), demonstrating the engraftment of ISL1+ CVP-derived cells 3 weeks post transplantation. Cell nuclei (blue) were stained with 4,6-diamindino-2-phenylindole (DAPI). Scale bar, 100 μm. (b) Representative images of xenograft showing engraftment of ISL1+ CVP-derived differentiated cells in the kidney capsule and myocardium. Vasculature network could be seen from the surface of the xenograft (kidney capsule), with numerous blood vessels carrying red blood cells (RBCs) as shown in the haematoxylin and eosin (H&E) and Masson stains (indicated with black arrows). Site of MI is marked by red arrow (lower panel). Connective tissues revealed by Masson stain (stained blue) were observed throughout the xenograft. Enlarged images show functional blood vessels carrying blood cells. RV, right ventricle; LV, left ventricle. Scale bar, 100 μm. (c) Representative immunofluorescent image of muscularized blood vessels in xenografts (both kidney capsule and myocardium). Rings of endothelial cells were stained positive for CD31 (stained red). The arteries were surrounded by additional ring of smooth muscle cells that were stained positive for α-SMA. Cell nuclei (blue) were stained with DAPI. Scale bar, 50 μm. (d) Graphical representation of the numbers and sizes of blood vessels observed in the xenograft per field (transplantation into myocardium after MI). Results were tabulated from 10 random fields of H&E stains of the xenografts. Bars, s.d. of n=3 sections. D, diameter. *P<0.05 and **P<0.001, evaluated by Student's t-test. (e) Large coronary-like artery developed from the transplanted ISL1+ CVP in the kidney capsule. Immunofluorescent staining of the section revealed an inner layer of endothelial cells (stained positive for CD31), which is surrounded by a thick layer of smooth muscle cells that were stained positive for both SMMHC and α-SMA. Connective tissue surrounding the artery was further depicted by the presence of fibrillin-1 (FBN1) as well as Masson stain (blue). Cell nuclei (blue) were stained with DAPI. Scale bar, 100 μm. Scale bar in enlarged image, 50 μm.