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. 2020 Mar 5;11:1195. doi: 10.1038/s41467-020-14764-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Schematic of NOG mouse ocular I/R experimental system for testing in vivo functionality of human primed DVP vs. N-DVP (modified from Park et al., 2014⁷). (left) Anatomical structures where I/R (anterior chamber) and human DVP and N-DVP cell injections (vitreous body) were performed (left panel). (right) Timeline for I/R injury surgery, human DVP injections (Day 0), human cell survival and engraftment analysis. b Human DVP survival at the superficial layer of murine retina at 3 weeks following injection of 50,000 DVP or N-DVP into the vitreous of I/R-treated NOG mouse eyes. Flat whole-mounted retinae were stained with antibodies for human-specific HNA (red), and tile scanned by confocal microscopic imaging (10x objective, 9 × 9 tiles). Shown are representative whole retinal images with HNA⁺ cells from primed DVP cell-injected (left panel) vs. N-DVP cell-injected (right panel) eyes. Scale bars = 500 μm. c Quantitation of HNA⁺ cells detected in the outer superficial layers of whole mount retinae following treatment of eyes with and without I/R, and injected with either primed DVP or N-DVP at (c) 4 weeks or (d) at 1, 3, and 4 weeks following DVP vs. N-DVP vs. control saline (PBS) injections, in eyes treated with and without I/R injury. Shown are the mean numbers from independent eye experiments of total HNA⁺ cells counted with imaging software per superficial layer of each whole-mounted retinae (whole field). **p < 0.01 (Mann-Whitney tests). e, f Human vascular engraftment into murine vessels. Whole-mounted retinae of I/R-injured eyes 2 weeks following DVP vs. N-DVP vs. PBS injections were immunostained with human CD34 (hCD34) to detect human endothelial engraftment. Antibodies for murine collagen type-IV (mCol-IV) were also employed to detect murine blood vessel basement membrane, and murine CD31 (mCD31) to detect murine endothelium. f The number of CD34⁺ human-murine chimeric vessels per 450 μm cross-section was quantitated via confocal microscopy and imaging software. Shown are results of independent measurements. ***p < 0.001 (unpaired t-tests).