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. 2021 Aug 5;11:15889. doi: 10.1038/s41598-021-95434-4

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© The Author(s) 2021

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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Donor cell integration induces tissue remodeling after ex vivo transplantation. (A) Representative confocal z-stack image of SM22⁺ (red) smooth muscle within control (non-transplanted) C57BL/6J colonic tissue at day-21. (Aⁱ) Orthogonal view

taken from the region of interest (ROI, cyan box) in A showing SM22⁺ smooth muscle in two uninterrupted layers. (B, C) Representative images of individual z-stacks at the longitudinal (B) and circular (C) muscle layers. (D) Representative confocal z-stack image of donor GFP⁺ (green) and SM22⁺ (red) smooth muscle 21-days after ex vivo transplantation. (Dⁱ&Dⁱⁱ) Orthogonal views taken from ROIs (cyan boxes) in (D) showing interruption of the longitudinal smooth muscle (Dⁱ) at the site of neurosphere engraftment. At sites away from the area of neurosphere engraftment SM22⁺ expression appears to demonstrate two uninterrupted smooth muscle layers (Dⁱⁱ). (E, F) Representative images of individual z-stacks at the longitudinal (E) and circular (F) muscle layers 21-days after ex vivo transplantation. (G) Representative image of individual z-stack, at the level of the circular muscle (taken from D), showing migration of GFP⁺ donor derived cells from the site of neurosphere engraftment 21-days after ex vivo transplantation. (H) Representative confocal z-stack image of Collagen IV (grey) in control (non-transplanted) C57BL/6J colonic tissue at day-21. (I) Representative confocal z-stack image of Collagen IV (grey) 21-days following ex vivo transplantation. Note the presence of Collagen IV expression at the site of neurosphere transplantation (cyan arrows) and apparent discontinuity of Collagen IV expression surrounding the presumptive neurosphere boundary (magenta dashed lines). (Iⁱ, Iⁱⁱ) Representative GFP channel (Iⁱ) and merged (Iⁱⁱ) confocal z-stack images showing GFP⁺ donor cells at the site of transplantation and within the tunica muscularis (arrows) having migrated away from the engrafted neurosphere.