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. 2019 May 6;9:6965. doi: 10.1038/s41598-019-43482-2

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© The Author(s) 2019

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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Optimisation of the immunosuppressive protocol for successful transplantation of mouse MNBs into rats. (a) Schematic of the experimental protocol. (b) Pre-transplanted MNB of the E13.5 mouse embryo. We observed immature nephrons at several developmental stages in the pre-transplanted MNs from the vesicles to the S-shaped bodies. (c) No TAC treatment. Fourteen days after transplantation, normal renal structures were not recognised due to severe inflammatory cell infiltration (n = 8 MNBs from two independent experiments). (d) Treatment with 0.3 mg/kg/day s.c. of TAC. Renal features remained, but glomeruli and tubule destruction and severe inflammatory cell infiltration were observed (n = 8 MNBs from two independent experiments). (e) Treatment with 2 mg/kg/day s.c. of TAC: the MNs showed normal renal features with minor inflammatory cell infiltration, but urine production was not confirmed (n = 8 MNBs from two independent experiments). (f) Treatment with 2 mg/kg/day s.c. of TAC + 5 mg/kg/day i.p. of MP: the MNs showed normal renal features, and what seemed to be urine production was confirmed in some specimens. The arrow indicates the embryonic bladder, which is filled with liquid. The arrowheads suggest urine production (n = 8 MNBs from two independent experiments). (g) Treatment with 2 mg/kg/day s.c. of TAC + 5 mg/kg/day i.p. of MP + 40 mg/kg/day p.o. of MMF. Few glomeruli remained, suggesting that MMF suppressed the development of MNs (n = 7 MNBs from two independent experiments). (h) The number of glomeruli per unit area was significantly higher in the 2 mg/kg/day of TAC group and the 2 mg/kg/day of TAC + 5 mg/kg/day of MP group than that in the other groups (n = 16, 16, 16, 16, 14 of MNs for each group, respectively). **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Error bars in bar plots represent the standard errors of the mean. (b–g) Left column: stereomicrographs of MNB (scale bars 500 µm), middle column: HE staining of MN at low magnification (scale bars 100 µm), right column: HE staining of MN at high magnification (scale bars 50 µm).