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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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Smad4 knockdown within Myh11-derived MSCs does not abolish induction of proliferative vitreoretinopathy following intravitreal injection of these cells within the murine OIR model. (A) A Luminex multiplex assay demonstrated no significant difference in active TGFβ1 protein concentration within the neural retina and vitreous of P14 wildtype (WT) mice and WT mice that underwent OIR (n = 5 unpaired eyes). Cropped Western blot from representative lanes of one gel (B) and densitometry quantification (C) demonstrated significant knockdown of Smad4 in Myh11-derived MSCs through mSmad4-shRNA adenovirus vectors (n = 6 biological replicates). Data are represented as mean ± standard error of mean (SEM). (D) Experimental design illustrating the intravitreal injection of Smad4-shRNA infected Myh11-derived MSCs versus Scramble-shRNA infected Myh11-derived MSCs at P12 following OIR injury, with subsequent harvest of injected retinas at P17. (E) Representative tile scan images of immunostained retinas revealed Col-IV pre-retinal matrix production (white outline in Merge panels) in eyes of P17 OIR mice intravitreally injected at P12 with passasge 6–8 Myh11-derived MSCs infected with either Ad-GFP-U6-mSmad4-shRNA or with Ad-GFP-U6-scramble. Fibrotic scar indicated by Col-IV is evident in both eyes regardless of Smad4 knockdown. Scale bar, 1000 µm. (F) No significant difference is found in fibrotic scar Col-IV matrix expression of the eyes intravitreally injected with Myh11-derived MSCs infected with Ad-GFP-U6-scramble adenovirus vectors (GFP+) as compared to Myh11-derived MSCs infected with Ad-GFP-U6-mSmad4-shRNA adenovirus vectors (GFP+) (n = 6 paired eyes). (G) No significant difference is found in fibrotic scar Col-IV matrix area normalized by the number of GFP+ cells found within this fibrotic scar. ****p < 0.0001. Data were analyzed using unpaired t test (A, C), or Wilcoxon test (F, G).