The Institutional Animal Care and Use Committee and the Safety Committee for Genetic Recombination Experiments of Jikei University School of Medicine approved the protocols for animal experiments (permission numbers 2021-017, 2019-013, R1-68). The experiments were conducted in accordance with the Guidelines for the Appropriate Conduct of Animal Experiments (2006) of the Science Council of Japan. Every effort was made to minimize animal suffering. Pregnant female C57BL/6JJmsSlc mice (B6 mice), pregnant female Sprague-Dawley-Tg (CAG-EGFP) rats (GFP rats), and mature male Sprague-Dawley rats (SD rats) were purchased from SLC (Shizuoka, Japan). Mature male NOD/Shi-scid, IL-2RγKO Jic mice (NOG mice) were purchased from CLEA (Tokyo, Japan).

Single cells from mouse and rat fetal kidneys were obtained following a method (10) that was a partial modification of a method reported previously (11). In brief, pregnant E13.5 B6 mice or E15.5 GFP rats were anesthetized via the inhalation of isoflurane (2817774, Pfizer, New York, NY, USA). Fetuses were harvested and the maternal mouse or rat was immediately euthanized by injecting pentobarbital (120 mg/kg; Kyoritsu Pharma, Tokyo, Japan). Fetuses removed from the mother’s womb were immediately decapitated and euthanized. Fetal kidneys were harvested from the decapitated fetuses under an operating microscope and collected in 1.5-ml tubes containing α minimum essential medium (MEMα; 12561-056, Thermo Fisher Scientific, Waltham, MA, USA). The tube was then centrifuged at 700 g for 3 min, the supernatant was removed, and 1 ml of accutase (AT104, Innovative Cell Technologies, San Diego, CA, USA) was dispensed. The sample was vortexed and incubated at 37°C for 5 min (repeated twice), and further manual pipetting and incubation was performed again for 5 min. The cell suspension was then centrifuged at 300 g for 5 min to remove the subsequent supernatant of accutase. The pellet was resuspended in a volume of 1 ml of MEMα with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% antibiotic-antimycotic (15240062, Thermo Fisher Scientific), and 10 µM Y2763 (257-00511, Wako, Osaka, Japan). In addition, the cells were passed through a 40 μm cell strainer (352340, Corning, NY, USA) to remove clumps of cells to obtain a single-cell suspension of mouse or rat cells. Cell counting of the single-cell suspensions was performed, and mouse and rat single-cell suspensions were placed on U-bottom 96-well low-cell-binding plates (174929, Thermo Fisher Scientific) in the cell ratios 6:0, 5:1, 3:3, 1:5, and 0:6 to a total of 2 × 10⁵ cells/well. Finally, the plates were centrifuged at 1,000 rpm for four min and incubated at 37°C in an incubator.

The single cells were confirmed to aggregate into spheroids by the next day (Day 1). After that, the medium containing Y27632 was removed, and the cells were cultured in medium containing MEMα, 10% FBS, and 1% antibiotic-antimycotic, with medium changes until Day 3. Spheroids were observed under an inverted microscope (Leica DMi1, Leica Microsystems, Wetzlar, Germany) or a fluorescence microscope (Olympus IX-71, Tokyo, Japan) and sampled on Day 4 or implanted under the renal capsule of NOG mice or SD rats. Specimens were whole-mount immunostained or frozen sectioned for fluorescent immunostaining or hematoxylin–eosin (HE) staining.

Renal organoids were transplanted under the renal capsule using a previously described method (10). The following is a brief description of the procedure. First, the recipient was anesthetized with isoflurane inhalation and a midline abdominal incision was made. The intestine was moved to the left or right side to expose the kidney, and the renal capsule in the lower part of the kidney was dissected at nearly 1 mm using a microshear. Then, the tip of the outer cylinder of the 22G surflo (SR-OT2225C, Terumo Corp, Tokyo, Japan) was cut at an angle. While the cut surface of the outer cylinder of the surflo was facing the renal parenchyma, it was inserted through the incision in the renal capsule to avoid damaging the renal parenchyma, and a small amount of saline water was placed under the renal capsule to detach a part of the renal capsule. After that, spheres in a 96-well plate were inhaled using the outer cylinder of the surflo with a syringe attached. The outer cylinder of the same surflo was inserted into the incision, and the renal organoids were transplanted under the renal capsule. The abdomen was then closed with a 5-0 thread. The recipients were mature male NOG mice or SD rats. Recipient SD rats received a small dose of an immunosuppressive drug (tacrolimus 0.3 mg/kg/day, Astellas Pharma, Tokyo, Japan), which was rejected when MNs of B6 mice were xenotransplanted into SD rats (12). At 14 days after transplantation, recipient NOG mice or SD rats were euthanized, and the transplanted renal organoids were subsequently collected and observed under a fluorescent stereomicroscope (Leica M205FA, Leica Microsystems, Wetzlar).

Renal organoid specimens for whole-mount immunostaining were fixed in 4% paraformaldehyde (163-20145, Wako) for 15 min at 4°C and then washed thrice with phosphate-buffered saline (PBS; 049-29793, Wako). Specimens were blocked for 1 hour at room temperature with 1% donkey serum (017-000-001, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), 0.2% skim milk (190-12865, Wako), and 0.3% Triton X-100/PBS (25987-85, Nacalai Tesque, Kyoto, Japan) and were subsequently incubated with primary antibodies overnight at 4°C. After washing thrice with PBS, the samples were incubated with secondary antibodies conjugated with Alexa Fluor 488, 546, and 647 for 1 hour at room temperature. Specimens were mounted with SlowFadeTM Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; S36973, Invitrogen, Carlsbad, CA, USA). Specimens were observed under a fluorescence microscope (LSM880 confocal, Carl Zeiss). Specimens of renal organoids to be cryosectioned were fixed in 4% paraformaldehyde in PBS for 60 min and dehydrated in 20% sucrose in PBS. Specimens were embedded in OCT compound (4583, Sakura Finetek, Tokyo, Japan) and 8-μm thick frozen sections were prepared. HE staining was performed according to the standard procedures for histological analysis. Antigen activation for immunofluorescence staining was performed in a warm bath at 70°C for 20 min. After blocking for 1 hour at room temperature, the sections were washed thrice with PBS and then incubated with primary antibodies overnight at 4°C. The sections were then washed thrice with PBS and incubated with secondary antibodies conjugated with Alexa Fluor 488, 546, and 647 for 1 hour at room temperature. Afterward, the sections were then washed thrice with PBS and mounted using SlowFadeTM Diamond Antifade Mountant with DAPI and observed under an all-in-one fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) or a fluorescence microscope (LSM880 confocal, Carl Zeiss). The primary antibodies used were as follows: chicken anti-GFP (ab13970, Abcam, Cambridge, MA, USA), rabbit anti-Six2 (11562-1-AP, ProteinTech, Rosemont, IL, USA), guinea pig anti-nephrin (GP-N2, Progen, Heidelberg, Germany), goat anti-megalin (sc-16478, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-E-cadherin (Ecad) (610181, BD, San Jose, CA, USA), rat anti-cytokeratin 8 (TROMA-I-C, DSHB, Iowa City, IA, USA), rabbit anti-platelet-derived growth factor receptor b (PDGFRb) (ab32570, Abcam), mouse anti-α SMA (A2547, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-CD31 (ab28364, Abcam), rabbit anti-NKCC2 (SPC-401D, StressMarq Bioscience), mouse anti-aquaporin 2 (sc-515770, Santa Cruz Biotechnology), rabbit anti- V-ATPase B1/2 (sc-55544, Santa Cruz Biotechnology), goat anti-GATA3 (AF2605, R&D Systems), and goat anti-renin (AF4277, R&D Systems).

Immunostained sections of frozen organoids were photographed with a fluorescence microscope. The total number overall and the number of GFP-positive cells of Six2-positive NPCs and GATA3-positive UBs in each cap mesenchyme were counted under the same magnification, and the percentage of GFP-positive NPCs and GFP-positive UBs (%) was calculated by dividing the number of GFP-positive cells by the total number of cells. Two randomly selected sections from the renal organoid specimen were photographed at the same magnification, and a total of six images of each were made for analysis. This work was performed by two researchers.

Immunostained sections of frozen organoids were photographed with a fluorescence microscope. The total number overall and the number of GFP-positive cells of GATA3-positive cells as collecting ducts, Ecad-positive and GATA3-negative cells as nephrons, and PDGFRb-positive cells as renal stroma were counted under the same magnification, and the percentage (%) of GFP-positive cells in each of the three lineages was calculated by dividing the number of GFP-positive cells by the total number of cells. Two randomly selected sections from the renal organoid specimen were photographed at the same magnification, and a total of six images of each were made for analysis. This work was performed by two researchers.

Immunostained sections of each frozen organoid were photographed with a fluorescence microscope. The number of CD3-positive cells per unit area (pcs/mm²) was calculated by counting the total number of CD3-positive T lymphocytes under the same magnification and dividing the total number of cells by the area. Two randomly selected sections from two specimens of renal organoids were photographed at the same magnification, making a total of six images each for analysis. This work was performed by two researchers.

HE-stained sections of organoid specimens of each cell ratio were photographed under the all-in-one fluorescence microscope to determine the number of glomeruli and area of organoids. The number of glomeruli per unit area (pcs/mm²) was then calculated by dividing the number of glomeruli by the area of the organoid. Two specimens from each group of renal organoids of each cell ratio were taken, and three randomly selected sections were photographed at the same magnification for a total of six images in each group for subsequent analysis. This measurement process was performed by two researchers.

Data were expressed as the mean ± standard error of the mean and analyzed using the Kruskal–Wallis test with a post-hoc test for comparison. All statistical analyses were performed using the Prism 8 software, and a p-value of <0.05 was considered significant.

Fetal kidneys of B6 mice and GFP rats were subjected to enzymatic treatment to obtain single cells. Single mouse cells were mixed with single rat cells in the same proportion to create chimeric cell aggregates via centrifugation on Day 0. On Day 1, chimeric spheroids of B6 mice and GFP rats were formed ( Figure 1A ). Next, we observed whether the nephrogenic niche of the interspecies chimeric renal organoids was reconstructed by whole-mount immunostaining on Day 3 ( Supplemental Figure 1 ). In the renal organoids of mice only, a cap mesenchyme was reconstructed in which six2-positive and GFP-negative mouse NPCs aggregated around cytokeratin 8 (CK8)-positive and GFP-negative mouse UBs ( Figure 1B , left). In GFP rat-only organoids, cap mesenchyme was reconstructed with six2-positive and GFP-positive rat NPCs aggregated around GATA3-positive and GFP-positive rat UBs ( Figure 1B , right). In the mouse-rat chimeric renal organoids, the cap mesenchyme was also reconstructed, and NPCs and UBs were composed of GFP-negative mouse cells and GFP-positive rat cells in a mosaic pattern ( Figure 1B , middle). In addition, immunostaining of frozen sections was used to verify the extent to which GFP-positive rat renal progenitor cells contributed to the cap mesenchyme. The total number of Six2-positive NPCs was 55.2 ± 12.1, the number of GFP-positive and Six2-positive NPCs was 18.0 ± 4.3, and the GFP-positive rate of NPCs was 34.5 ± 6.7%. The total number of GATA3-positive UB cells was 15.5 ± 1.7, the number of GFP-positive and GATA3-positive UB cells was 8.0 ± 2.8, and the GFP-positive rate of UB was 45.6 ± 14.6% ( Figure 1C ).

Chimeric renal organoids—a mixture of single cells from the fetal kidneys of B6 mice and GFP rats in the same proportion—were transplanted under the renal capsule of adult NOG mice—an in vivo environment—for further maturation ( Figure 2A and Supplemental Figure 2 ). The specimens were collected on day 14 because the chimeric organoids gradually became disrupted by their own hydronephrosis after long-term transplantation beyond day 14. At the time of retrieval after 14 days, the chimeric renal organoids expressed GFP and were invaded by GFP-negative blood vessels derived from the recipient mice ( Figure 2B , yellow arrowhead). Cryosections of the chimeric renal organoids showed extensive GFP expression, and HE staining revealed luminal structures ( Figure 2C ). Moreover, fluorescence immunostaining of the cryosections showed that the whole chimeric renal organoids comprised GFP-positive rat cells and GFP-negative mouse cells, with Ecad-positive tubules ( Figure 2D ), nephrin-positive glomeruli, and PDGFRb-positive renal stroma ( Figure 2E ). In GATA3-positive and Ecad-positive collecting ducts comprising GFP-positive rat cells and GFP-negative mouse cells were also observed ( Figure 2F ). Aquaporin 2-positive, GATA3-positive, GFP-positive rat principal cells, ( Figure 2G ) and V-ATPaseB1-positive, GATA3-positive, and GFP-positive rat intercalated cells ( Figure 2H ) were observed, forming luminal structures with GFP-negative mouse cells. Ecad-positive and GFP-positive rat distal tubules were connected to the GFP-negative and CK8-positive mouse collecting ducts ( Figure 2I ). An NKCC2-positive loop of Henle comprising GFP-positive rat cells and GFP-negative mouse cells was observed ( Figure 2J ). A megalin-positive and GFP-positive proximal tubule forming a lumen with GFP-negative mouse cells was also observed ( Figure 2K ). In the nephrin-positive glomeruli, podocytes comprised GFP-positive rat cells and GFP-negative mouse cells and PDGFRb-positive mesangial cells comprised GFP-positive rat cells and GFP-negative mouse cells ( Figure 2L ). PDGFRb-positive interstitial fibroblasts also comprised GFP-positive rat cells and GFP-negative mouse cells ( Figure 2M ). Furthermore, α SMA-positive vascular pericytes comprising GFP-positive rat cells were observed ( Figure 2N ). In addition, as proof of organoid functionality, we observed GFP-positive renin-producing cells with endocrine functions ( Figure 2O ). We also examined the contribution of GFP-positive rat cells to each of the three lineages by counting the cells that were immunostained for representative cell markers. The percentage of GFP-positive cells in GATA3-positive collecting ducts was 66.1 ± 9.3%, that of GFP-positive cells in the tubules of Ecad-positive and GATA3-negative nephrons was 37.1 ± 9.4%, and that of GFP-positive cells in PDGFRb-positive stroma was 44.2 ± 6.7% ( Figure 2P ).

To investigate whether chimeric renal organoids reduce rejection compared with xenograft organoids, renal organoids were prepared in vitro by mixing cells from the fetal kidneys of B6 mice and SD rat-based GFP rats in various ratios (6:0, 5:1, 3:3, 1:5, and 0:6) and transplanted into the renal capsule of adult SD rats. The recipients were subjected to mild immunosuppression (tacrolimus 0.3 mg/kg/day) and organoids of the respective cell ratios were retrieved at 14 days after transplantation ( Supplemental Figure 3 ). At the time of retrieval, 6:0 xenograft mouse renal organoids and 5:1 and 3:3 chimeric renal organoids were white and swollen, whereas 1:5 chimeric renal organoids and 0:6 allogeneic rat renal organoids were not swollen, showing vascular invasion from the recipient rat and strong GFP expression ( Figures 3A, B ). HE staining of the same tissue showed that the 6:0 xenograft mouse renal organoids and the 5:1 and 3:3 chimeric renal organoid tissues were thick and swollen with strong inflammatory cell infiltration and few glomeruli ( Figures 3C, D, F ). However, the 1:5 chimeric renal organoids, in which the cell ratio of the same rat as the recipient was increased, showed mild infiltration of inflammatory cells and mild abolition of glomeruli by inflammatory cells ( Figures 3C, D, F ). Furthermore, inflammatory infiltration was evaluated by immunostaining for CD3. Infiltration of CD3-positive cells was observed mainly in areas composed of xenogeneic, GFP-negative mouse cells, whereas areas generated from GFP-positive rat cells showed less infiltration of CD3-positive cells, and the remaining glomeruli were composed mainly of GFP-positive rat cells ( Figure 3E ). The number of CD3-positive cells per unit area was not significantly different in the 1:5 chimeric renal organoids compared to the control 0:6 allogeneic rat renal organoids, but the number of CD3-positive cells was significantly increased in the 6:0 xenograft renal organoids and the 5:1 and 3:3 chimeric renal organoids ( Figure 3G ).