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. 2020 Sep 25;10:15808. doi: 10.1038/s41598-020-72875-x

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Within the murine OIR model, intravitreal injected Myh11-derived MSCs in the vitreous gel exhibit a myofibroblast phenotype, while endogenous, retinal Myh11+ mural cells remain in a perivascular position. (A) Immunostained Myh11-derived MSCs lacked expression of Col-IV in vitro, however, (B) immunostained retinas revealed intravitreal injected Myh11-derived MSCs expressed Col-IV in the vitreous gel, which formed a dense, fibrotic pre-retinal membrane in murine OIR eyes. Scale bars, 200 µm. (C) Intravitreal injected passage 3–5 Myh11-derived MSCs expressed αSMA+ stress fibers and Col-IV, and (D) Myh11-derived MSCs have reduced expression of Myh11 following injection (arrow) compared to the endogenous Myh11 expressed in retinal mural cells. DAPI stained nuclei of underlying retinal ganglion cells in addition to injected MSCs. Scale bars, 100 µm. (E) Experimental design where tamoxifen is delivered postnatal day 1–3 Myh11-tdTomato mice to induce expression of tdTomato in Myh11+ mural cells. Induced mice are then exposed to hyperoxia from postnatal day 7–12 to cause OIR injury, with retinas harvested at P17 to determine cell fate of endogenous, retinal Myh11+ mural cells. (F) At P17, endogenous, retinal Myh11+ mural cells resided on Col-IV+/CD31+ vessels, with αSMA expression higher in vSMCs (arrow) than PCs (asterisk). Scale bar, 100 µm. (G) Myh11+ mural cells remained on vessel with no vSMCs-PCs found off vessel. Scale bar, 100 µm. (H) Neither retinal vSMCs or PCs extended processes from CD31 tip cells (arrow) at the leading front of the regenerating retinal microvasculature. Scale bar, 25 µm. Immunohistochemistry images represent fields of view that were sampled based on the presence of eYFP and tdTomato expression within culture and tissue. Images are also representative of at least three biological replicates or animals.