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. 2017 Aug 31;7:9891. doi: 10.1038/s41598-017-09927-2

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© The Author(s) 2017

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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The subrenal capsule can support the development of the transplanted neonatal small intestine to complete maturation. (a,a’,h,h’) H&E staining for transplanted small intestine harvested from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days post-transplantation. (b,b’,i,i’) Immunostaining for lysozyme (red, Lys) on sections of transplanted small intestine derived from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days after transplantation (green, Lgr5). (c,c’,j,j’) Immunostaining for Ki-67 (brown), as a marker for cell proliferation, on sections of transplanted small intestine derived from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days post-transplantation. (d,k) Periodic acid-Schiff (PAS) staining for transplanted small intestine harvested from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days after transplantation. (e,l) Alkaline phosphatase staining of transplanted small intestine harvested from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days after transplantation. (f,m) Immunostaining of transplanted intestinal tissue revealing cell maturation based on sucrose-isomaltase (SIM) expression. (g,g’,n,n’) Immunostaining for mMECA-32 (brown) (g’,n’: black arrows), as a marker of vasculature ingrowth, on sections of transplanted small intestine derived from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days after transplantation. Higher magnification images (a’,b’,c’,g’,h’,i’,j’,n’) of the corresponding boxed area are shown on the right side of the respective images. White dashed lines indicate the boundary between the epithelium and lamina propria. Scale bars: orange, 50 μm; red, 20 μm; white, 10 μm. (o) The crypt length of transplanted small intestine harvested from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days after transplantation. Error bars indicate standard deviation. **p < 0.001. D; Days after transplantation. The numbers of crypts analyzed were as follows: D5; n = 62 in 10 transplants, D7; n = 47 in 8 transplants, D10; n = 50 in 7 transplants, and D14; n = 57 in 11 transplants derived from 4 mice (D5), 2 mice (D7), 2 mice (D10), and 3 mice (D14), respectively. (p) The villus length of transplanted small intestine harvested from P0 Lgr5 ^{EGFP-IRES-CreERT2/+} mice at the indicated days after transplantation. Error bars indicate standard deviation. **p < 0.001. D; Days after transplantation. The number of villi analyzed were as follows: D3; n = 48 in 8 transplants, D5; n = 66 in 10 transplants, D7; n = 44 in 8 transplants, D10; n = 50 in 7 transplants, D14; n = 49 in 11 transplants derived from 2 mice (D3), 4 mice (D5), 2 mice (D7), 2 mice (D10), and 3 mice (D14), respectively. (q) The number of Lgr5-EGFP⁺ cells per inter-villus region or crypt. Error bars indicate standard deviation. D; Days after transplantation. The numbers of crypts analyzed were as follows: D3; n = 55 in 8 transplants, D5; n = 40 in 10 transplants, D7; n = 41 in 8 transplants, D10; n = 45 in 7 transplants, and D14; n = 40 in 11 transplants derived from 2 mice (D3), 4 mice (D5), 2 mice (D7), 2 mice (D10), and 3 mice (D14), respectively.