Urine excretion strategy for stem cell-generated embryonic kidneys

Significance

Worldwide, the number of patients with end-stage renal disease requiring renal replacement therapy is increasing because of the shortage of donor organs. We have successfully generated functional kidneys from human stem cells using the organogenic niche method. However, for these kidneys to have clinical application, a urinary excretion pathway is necessary. Using pigs, we demonstrated our stepwise peristaltic ureter system, showing that it resolves important problems regarding the construction of the urine excretion pathway and the long-term growth of the stem cell-generated embryonic kidneys.

Abstract

There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal’s ureter to the cloacal-developed bladder, a technique we called the “stepwise peristaltic ureter” (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney.

In recent years, the number of patients with chronic kidney disease has been increasing worldwide. Because of organ donor shortages, the number of patients with end-stage renal disease (ESRD) requiring renal replacement therapy is increasing also, and these patients are at increased risk of cardiovascular disease and death (1, 2). Thus, ESRD is a major clinical problem. Recently, remarkable advances have been made in stem cell-based therapies for organ generation, and many studies have demonstrated the possibility of using stem cells to generate neo-kidneys. Nevertheless, the kidney remains one of the most difficult organs to reconstruct de novo because of its delicate and complicated architecture.

We recently generated a functional kidney de novo using the organogenic niche method (3–5). This method involved microinjecting human mesenchymal stem cells (hMSCs) into the budding region of a rat embryo. Histologically, the injected hMSCs formed a mature kidney structure, including glomerular podocytes and tubular epithelial cells (3). Histologic examination of differentiated metanephroi after transplantation into rat omenta showed they consisted of human nephrons invaded by the vascular system of the recipient. This observation indicated that the glomerular endothelial cells had originated from the recipient (4). The neo-kidney produced urine, erythropoietin in the presence of anemia (6), and renin in the presence of hypotension (7). However, the nascent kidney ultimately developed hydronephrosis and did not grow in size because it lacked a urine excretion channel (4).

In other studies, we (8), along with another group (9) generated a whole organ de novo from exogenic pluripotent cells by using the blastocyst complementation method, which is one of the most promising methods in this field of research. However, the generated organ’s vascular system was a chimeric tissue derived from both recipient animal cells and injected exogenic cells. A vascular system originating from the exogenic cells might become a target of the host’s immune response. To avoid this problem, an embryonic kidney that has not yet developed a vascular system must be transplanted into a host.

If these strategies are to be applied to generating human kidneys from stem cells, the generated kidneys must have urine excretion channels. Here, we demonstrate the generation of such a channel in syngeneically transplanted embryonic pig metanephroi using the stepwise peristaltic ureter (SWPU) system. Briefly, we transplanted metanephroi along with the cloaca (from which the bladder developed) into host animals and then connected the host animal’s ureters to the developed bladder at an appropriate time (Fig. S1A). Allowing the kidney to grow large is another important issue; therefore, we used the pig, a relatively large animal, to provide a better test of the method’s feasibility for clinical application (10).

Fig. S1.

A urine excretion strategy for neo-kidneys. (A) SWPU system. Metanephroi with bladders grown from cloacas were transplanted into host animals. After transplantation, we removed the left native kidney and connected the left native ureter to the cloaca-grown bladder. (B) Conventional connection to the recipient ureter. After matanephroi transplantation, rats with transplanted metanephroi underwent a single end-to-end anastomosis to connect the ureter of the metanephros to the host ureter.

Results

Syngeneic Transplantation of Pig Metanephroi.

Cloned pig fetuses at embryonic day (E) 30 were recovered from an excised uterus (Fig. 1A and Fig. S2). Primordial pig metanephroi were dissected under a stereomicroscope (Fig. 1B), and some were cryopreserved. A preliminary experiment confirmed the absence of significant differences in the growth of vitrified and nonvitrified pig metanephroi transplanted into mice (Fig. S3). We then implanted pig metanephroi in the omenta of anesthetized syngeneic host pigs (Fig. 1C). All transplanted metanephroi differentiated successfully into mature kidneys, growing to about 5–7 mm in length 3 wk after transplantation (Fig. 1 D and E). Histopathologic examination of the transplant-grown metanephroi showed mature glomeruli and renal tubules (Fig. 1 F and G). Five weeks after transplantation, the implanted metanephroi had grown to more than 1 cm in length and had developed ureters, which retained urine produced by the metanephroi (Fig. 1 H and I). Some recipient vessels were integrated into the metanephroi, and the differentiated metanephroi maintained their glomeruli and renal tubules, with little interstitial tissue hemorrhage (Fig. 1 J and K). Eight weeks posttransplantation, the metanephroi had grown to about 3 cm in length; however, urine production had led to dilated ureters, resulting in hydronephrosis (Fig. 1 L and M). Histologic analysis indicated thinning of the cortex, interstitial fibrosis (Fig. 1N), and well-differentiated metanephroi ureters including both muscular and epithelial layers (Fig. 1O). These results suggest that a urine excretion channel is indispensable for the development of implanted metanephroi.

Fig. 1.

Syngeneic transplantation of metanephric primordia in pigs. (A) Cloned pig embryos at E30. (B) Cloned pig metanephroi removed from the embryos. (Scale bar: 2 mm.) (C) Metanephroi are syngeneically transplanted into the omenta of the recipient pigs. (D and E) Three weeks after transplantation, transplanted metanephroi are 5 mm in size. (F and G) Histopathologic analysis with Masson’s trichrome staining reveals that at 3 wk metanephroi have cortex tissue that consists of glomeruli and renal tubules. (H and I) Five weeks after transplantation, transplanted metanephroi are more than 1 cm long (H) and exhibit ureters (I). (J and K) Masson’s trichrome staining shows that at 5 wk transplanted metanephroi maintain glomerular structure and have little hemorrhage in the interstitial tissue. (L and M) Eight weeks after transplantation, blood vessels from the omentum are integrated into the metanephros, which is about 3 cm long, but the metanephros developed hydronephrosis. (N and O) Thinning of the cortex and interstitial fibrosis of metanephroi (N) and good differentiation of the metanephroi ureters, including muscular and epithelial layers (O) are observed in sections stained with Masson’s trichrome. BV, blood vessel from recipient’s omentum; C, cortex; M, medulla; U, ureter from transplanted metanephros.

Fig. S2.

Syngeneic transplantation of pig metanephroi. Cloned E30 pig metanephroi are dissected and implanted in the omenta of syngeneic, adult cloned gilts. Three, five, and eight weeks later, the recipient pigs are killed. POD, postoperative day.

Fig. S3.

Comparison of the differentiation potential of vitrified and nonvitrified pig metanephroi. Vitrified and nonvitrified pig metanephroi primordia (Pig MN) were transplanted to the omentum or abdominal wall of NOD/SCID mice (CLEA Japan). Vitrified (Vit) and nonvitrified pig metanephroi with bladders developed from cloacal transplantation (Pig CL) also were transplanted into NOD/SCID mice. Two weeks after transplantation, mice were killed. There are no significant differences in growth between vitrified and nonvitrified pig metanephroi. There also are no significant difference in growth between metanephri grown from MN transplantation and metanephroi grown from cloacal transplantation. Gl, glomerulus; Vit, vitrified. (Scale bars: 200 μm.)

Comparison Between Metanephros and Cloacal Transplantation In Rats.

In rats, metanephroi developed in the same way, regardless of whether the cells had come from the cloaca, permitting development of a bladder (the MNB group), or from metanephric primordia (the MN group) (Fig. 2 and Fig. S4). In both groups, the organ weights increased but peaked at 4 wk posttransplantation; there were no significant differences between the MN and MNB groups in the weights of the developed organs (Fig. 2B). However, renal tubular dilation was observed at 3 wk posttransplantation in the MN group (P < 0.05) (Fig. 2 A and C) and had progressed at 4 wk after transplantation (P < 0.0005) (Fig. 2C). Conversely, renal tubular dilation was not observed in the MNB group. Histopathologic examination showed that at the time the animals were killed the tubular lumina dilation, interstitial fibrosis, and reduction of glomerular numbers were more pronounced in the MN group than in the MNB group (Fig. 2 A and C–E); the vesicoureteral junction was better differentiated in the MNB group (Fig. S5); urine volume produced from the metanephroi was significantly larger in the MNB group than in the MN group (P < 0.05) (Fig. 2G); and urine levels of urea nitrogen (UN) and creatinine (Cr) were higher in the MNB group than in the MN group (P < 0.05) (Fig. 2 H and I). These findings indicate that MNB transplantation is superior to MN transplantation with regard to both kidney development and urine production.

Fig. 2.

Comparison of MN and MNB transplantation in rats. (A) Histopathologic analysis of differentiated metanephroi using Masson’s trichrome staining. Renal tubular dilation and interstitial fibrosis are more prominent in the MN group than in the MNB group, and the glomerulus count is lower. (Scale bars: 400 μm.) (B) There are no significant differences in the weights of either the metanephroi or bladders between the MN and MNB groups. (C) Renal tubular dilation is observed, beginning at 3 wk after transplantation, in the MN group and progressed by 4 wk after transplantation. However, renal tubular dilation is not observed in the MNB group at 4 wk after transplantation. (D) Interstitial fibrosis of metanephroi at sacrifice is more severe in the MN group than in the MNB group (P < 0.0005). (E) The glomerulus count of metanephroi at sacrifice is higher in the MNB group than in the MN group (P < 0.05). (F) Differentiated metanephroi in the MN and MNB groups 4 wk after transplantation. (G) Urine volume at sacrifice is significantly larger in the MNB group than in the MN group (P < 0.05). (H and I) Urea Cr (H) and UN (I) excretion is higher in the MNB group than in the MN group (P < 0.05).

Fig. S4.

E15 rat metanephroi and cloacal tissue were removed under stereomicroscopic guidance. In MNB groups 1–4 (n = 20), cloacas were implanted in the para-aortic area of recipient rats. In MN groups 1–4 (n = 16), metanephroi and bladder primordia were implanted in the para-aortic area of recipient rats after the ureter of the metanephros was cut. Ten days after surgery, rats in MN group 1 (n = 4) and MNB group 1 (n = 4) were killed. Two weeks after surgery, rats in MN group 2 (n = 4) and MNB group 2 (n = 4) were killed. Three weeks after surgery, rats in MN group 3 (n = 4) and MNB group 3 (n = 7) were killed. Four weeks after surgery, rats in MN group 4 (n = 4) and MNB group 4 (n = 5) were killed. POD, postoperative day.

Fig. S5.

Histopathologic analysis of the vesicoureteral junction in differentiated cloacas. The vesicoureteral junction is well-differentiated in the MNB group. The continuity of the cloaca-differentiated ureter and bladder is shown in serial sections (A–I). B, bladder; M, metanephros; U, ureter.

SWPU System.

We created a urinary excretion channel in rats using the SWPU system (Fig. S2A). E15 rat cloacas were transplanted into the para-aortic area of anesthetized rats. Four weeks after transplantation, we connected each rat’s left ureter to the newly developed bladder under stereomicroscopic guidance (Fig. S6). Seven to eight weeks after cloacal transplantation (3–4 wk after ureter–bladder anastomosis), urine from the bladder grown from the cloacal transplant was discharged continuously from the connected recipient ureter (Movie S1). I.v. urography showed that contrast medium appeared in the transplant-grown bladder after injection and then passed to the recipient ureter and bladder over time (Fig. 3A). UN and Cr levels were much higher in the urine from the transplant-grown bladder than in the sera of recipient rats (Fig. 3 F and G), suggesting that a bladder developed from a transplanted cloaca has the potential to collect urine within 8 wk after transplantation. Histopathologic analysis revealed successful anastomosis between the transplant-grown bladder and the recipient ureter (Fig. 3 B–D); even 8 wk after transplantation, the cloaca maintained mature renal structures, such as glomeruli and renal tubules (Fig. 3E). On the other hand, metanephroi that underwent anastomosis between the host and metanephroi ureters showed severe renal pelvis dilation and thinning of the cortex, resulting in hydronephrosis (Fig. S7). Furthermore, the SWPU system significantly prolonged the lifespan of anephric rats compared with controls (Fig. 3H and Fig. S8). The system allowed continuous discharge of urine from a transplant-grown bladder into a recipient bladder via the recipient ureter, thus providing a urinary excretion channel for the generated kidney; such discharge is difficult to achieve conventionally.

Fig. S6.

Rat metanephroi with bladders developed after cloacal transplantation using the SWUP system. E15 rat cloacas were removed and transplanted into recipient rats. Four weeks later, the left native kidney was removed, and the recipient left ureter was connected to the bladder grown after cloacal transplantation. Three or four weeks after this surgery, the developed cloaca was removed. POD, postoperative day.

Fig. 3.

SWPU system. (A) I.v. urography, using CT, shows that contrast medium appears in the cloaca-grown bladder after injection and then passes into the recipient ureter over time. (B and C) Histopathologic analysis using H&E staining shows successful anastomosis between the cloaca-grown bladder and recipient ureter. (D) Uroplakin III staining reveals the continuity of the transitional epithelium at the point of anastomosis. (E) The cloaca-grown kidney exhibits mature renal structures such as glomerular and renal tubules 8 wk after transplantation. (F and G) UN (F) and Cr (G) levels are much higher in the urine produced from the cloaca-grown kidney than in the sera of recipient rats. (H) Kaplan–Meier survival curve for rats with SWPU (continuous line, n = 14) and anephric control rats (dotted line, n = 7). Median survival of control rats with no native renal mass is 69.50 h. Animals transplanted with the SWPU system (median survival 85.38 h) survived longer than control animals (P < 0.05). A, anastomosis between cloaca-grown bladder and recipient ureter; B, cloaca-grown bladder; BL, urine in recipient bladder; CL, urine in cloaca-grown bladder; Gl, glomerulus; M, metanephros; Tu, renal tubules; U, recipient ureter. (Scale bars: 400 μm in B; 200 μm in E.)

Fig. S7.

Rat metanephros transplantation using uretero–ureterostomy. (A) E15 rat metanephroi were removed and transplanted into recipient rats. Four weeks later, the left native kidney was removed, and the recipient left ureter was connected with the metanephros-grown ureter. Three weeks after this surgery, the developed metanephros was removed. POD, postoperative day. (B) Seven weeks posttransplantation, the metanephros had grown to about 5 mm in length. (C–E) Histology of control rats undergoing uretero–ureterostomy reveals thinning of the cortex and severe dilation of renal pelvis, renal tubule, and Bowman’s capsule compared with rats subjected to the SWPU system, resulting in hydronephrosis. M, metanephros; RP, renal pelvis of metanephros; U, recipient ureter. (Scale bars: 1 mm in A–C; 200 μm in D and E.)

Fig. S8.

Survival of rats transplanted with the SWPU system. The E15 rat metanephroi and bladder tissue developed from the cloacal transplants were removed under microscopic guidance and were implanted in the para-aortic areas of recipient rats (n = 21). Four weeks after transplantation, rats in the SWPU group (n = 14) underwent left nephrectomy and received an anastomosis between the bladder grown from the cloacal transplant and recipient’s left ureter. Rats in the control group (n = 7) underwent left nephrectomy only. Eight weeks after transplantation, all rats underwent right nephrectomy. Rat life spans were measured from the time of right nephrectomy.

Syngeneic Cloacal Transplantation in Pigs Using the SWPU System.

First, we transplanted pig cloacas into syngeneic cloned pigs (Fig. 4A). Cloacas transplanted into omenta continued to develop in the same way as in rats, producing urine 3 wk after transplantation (Fig. 4B). Produced urine was retained in the transplant-grown bladder at 5 wk after transplantation (Fig. 4C). Next, we created a urinary excretion channel in cloned pigs using the SWPU system (Fig. S1A). E30 pig cloacas were transplanted into the parasplenic artery area of anesthetized pigs. Four weeks later, we connected the left ureter to the transplant-grown bladder (Fig. 4 D–F and Fig. S9). During this period, the levels of UN, Cr, and potassium (K) were much higher in the urine from the transplant-grown cloacas than in the sera of recipient pigs (Fig. S10). Histopathologic examination of the transplant-grown cloacas showed mature glomeruli and renal tubules (Fig. 4G). Cloned pigs were killed 8 wk after cloacal transplantation (4 wk after ureter–bladder anastomosis). At that time the metanephroi differentiated from cloacas maintained nephron structures similar to the structures seen at 4 wk posttransplantation (Fig. 4 G–I). At 8 wk posttransplantation the dilation of the tubular lumina and interstitial fibrosis was lower in the metanephroi of pigs in which the SWPU system had been applied than in those in which it was not applied (Fig. 1 N and O). These results suggest that the creation of a urinary excretion channel, using the SWPU system, permitted the transplanted cloacas to continue to develop for a long time. Thus, at least in pigs, the system is useful for creating urinary excretion channels and for increasing the size of the stem cell-generated embryonic kidneys.

Fig. 4.

Syngeneic transplantation of pig cloaca using the SWPU system. (A) Metanephroi (M) with bladders (CLs) from an E30 pig embryo. (B) Pig cloaca syngeneically transplanted into pig omentum, 3 wk after transplantation. Liquid is retained in the bladder grown from the transplanted cloaca. (C) Pig cloaca transplanted into pig omentum, 5 wk after transplantation, shows two metanephroi and a liquid-filled bladder. (D and E) Anastomosis between a bladder grown from a cloacal implant and a recipient ureter. Four weeks after cloacal transplantation, we connected the recipient’s left ureter to the bladder grown from the cloacal implant. (F) One day after ureter–bladder anastomosis, blood vessels from the recipient’s splenic artery are integrated into the structures grown from the cloacal implant. (G and H) H&E staining of a differentiated cloaca 4 wk after transplantation. (I) H&E staining of a differentiated cloaca 8 wk after transplantation. The kidneys exhibit structures such as glomeruli and renal tubules. B, bladder grown from a cloacal transplant; BV, blood vessel from recipient’s splenic artery; Gl, glomerulus; M, metanephros; S, spleen of recipient pig; Tu, renal tubules; U, recipient’s ureter. (Scale bars: 200 μm.)

Fig. S9.

Syngeneic metanephroi and bladder transplantation in pigs using the SWPU system. Cloned E30 pig cloacas were implanted into syngeneic, adult cloned pigs. Four weeks after implantation, recipient pigs underwent the SWPU system. One day or four weeks after this surgery, the developed cloacas were removed for examination.

Fig. S10.

Biochemical analysis of urine from metanephroi and bladders grown from cloacal transplants. Four weeks after transplantation, the levels of UN, Cr, and K were much higher in the urine from the bladders grown from cloacal transplants than in the sera of recipient pigs (P < 0.01, P < 0.05, P < 0.05, respectively). CL, urine in bladders grown from cloacal transplants.

Discussion

This report describes the construction of a urine excretion pathway for stem cell-generated embryonic kidneys that involved connecting the recipient ureter with a bladder grown from a transplanted embryonic cloaca. After cloacal transplantation, several vessels from the recipient animal were integrated into the transplanted cloaca. Thereafter, the metanephroi of the cloaca continued to develop and started to produce urine, as previously reported (3–7, 11). The produced urine was excreted into the cloacal bladder, via the cloaca’s ureter, by peristalsis. In both pigs and rats, urine collected in transplant-grown bladders and was discharged continuously by the peristaltic movements of the recipient ureter, preventing the transplant-grown cloaca from developing hydronephrosis. Eight weeks after transplantation, the concentration of UN and Cr were still much higher in the cloacal urine than in the sera of recipient rats (Fig. 3 F and G), suggesting that the SWPU system permits the transplanted cloaca to enlarge and replace kidney function in the recipient animals, an ability that, to our knowledge, has never before been demonstrated.

Previous studies described the direct connection of a recipient ureter with the ureter of a transplant-grown metanephros (uretero–ureterostomy) to create a urine excretion channel for a neo-kidney (Fig. S1B) (11–13), prolonging the short-term survival of anephric rats (12, 13). However, our SWPU system is more efficient than previous methods in many ways. First, cloacal transplantation is superior to metanephroi transplantation in preventing hydronephrosis. Our study indicated that transplanted metanephroi began urine production 3 wk posttransplantation and tended to develop hydronephrosis 4 wk after transplantation, suggesting that this method leads to renal insufficiency (Fig. 2A). Persistent urine discharge into the transplant-grown bladder seems to extend the time before hydronephrosis develops. Second, the SWPU system is more effective than previous methods in allowing sustainable growth and maturation of the kidney. During kidney development, sustained excretion of urine from the metanephroi is caused by peristalsis of the ureter primordia; ureter primordia obstruction has been suggested to result in dysplastic metanephroi (14). This study actually shows that the number of glomeruli and urine volume are larger following cloacal transplantation than after metanephroi transplantation. Third, the SWPU system can join two metanephroi at one time, whereas connecting the recipient ureter to two metanephroi ureters is difficult. Because previous studies showed that the total mass of the developed metanephros correlated with the duration of anephric rat survival (13), the SWPU system is thought to be more effective than conventional uretero–ureterostomies in improving survival time. Fourth, connecting the recipient ureter with a bladder grown from a cloacal transplant is easier than uretero–ureterostomy because the recipient ureter is very large compared with the metanephroi ureters, and the urine-expanded cloacal-grown bladder is either larger than or similar in size to the recipient ureter.

In future studies, we will regenerate cloacas from human stem cells in pig embryos using the organogenic niche method or blastocyst complementation method and then will transplant the cloaca into a human. The cloaca is expected to develop in the human, creating its own human-derived vascular system (15, 16). Then we will construct a urinary excretion channel using the SWPU system. However, for these strategies to work, kidney- and ureter-deficient pigs are required to avoid forming chimeric tissues. Mouse embryos lacking both Pax2 and Pax8 are unable to form metanephroi and ureters (17). Therefore, we plan to establish similar kidney- and ureter-deficient pigs to ensure that the ureter of the neo-kidney originates from injected human stem cells.

We have demonstrated that the SWPU system may provide the means to construct a urinary excretion pathway for stem cell-generated embryonic kidneys. The creation of such a pathway is one of the most important problems to be overcome in the de novo generation of whole kidneys, and the solution of this problem represents a significant advance in the field.

Materials and Methods

Animals.

Adult male Lewis rats were purchased from Sankyo Lab Service Corporation and CLEA Japan. Pairs of animals were kept in cages and allowed free access to food and water. Crossbred gilt pigs (Hypor Japan) were used as recipients of somatic cell nuclear transfer (SCNT) embryos for producing cloned pigs. The pigs were maintained in a semi-windowless facility with a controlled temperature (15–30 °C) and received a standard porcine diet twice daily and water ad libitum. All experimental procedures were approved by the committees for animal experiments and the ethics committees of Jikei University, Meiji University, and Kitasato University.

Experimental Protocols.

Experiment 1.

We generated cloned E30 pig fetuses from a line of female fetal fibroblast cells using somatic cell cloning technology, as described previously (8). Metanephroi were dissected from the cloned fetuses under a stereomicroscope (Fig. 1B) and were implanted in the omenta of syngeneic adult cloned gilts (Fig. 1C). Three, five, and eight weeks later, the recipient pigs were killed under general anesthesia induced using injected pentobarbital and inhaled isoflurane, and the transplanted metanephroi were recovered (Fig. S2).

Experiment 2.

Ten-week-old Lewis rats were divided into eight groups (Fig. S4). Rats in MNB groups 1–4 (n = 20) were implanted with cloacas in the para-aortic area. Rats in MN groups 1–4 (n = 16) were implanted with metanephroi, along with the bladders, after the metanephroi ureters in the para-aortic area were cut. Rats in both the MNB and MN groups were killed 10 d (group 1), 2 wk (group 2), 3 wk (group 3), and 4 wk (group 4) after transplantation. All of the developed metanephroi and bladders were removed at the time the animals were killed. Any metanephroi-produced urine that had collected in the bladders or ureters was extracted using a microneedle, and the volumes were measured.

Experiment 3.

E15 rat cloacas were removed and transplanted in the para-aortic area of 9-wk-old, anesthetized recipient rats. Four weeks after transplantation, we removed the left native kidney and connected the left ureter to the bladder of the transplanted cloaca, under microscopic guidance (Fig. S6). Three or four weeks after this surgery (7 or 8 wk after transplantation), the rats were subjected to computed tomography (CT) scans, and the developed cloacas were removed. To analyze the life span of SWPU system-treated rats, E15 rat cloacas also were implanted into the para-aortic areas of 9-wk-old Lewis rats (n = 21). Four weeks after transplantation, the SWPU group (n = 14) underwent left nephrectomies and received anastomoses between the bladders grown from cloacal transplants and the recipient left ureters. Rats in the control group (n = 7) underwent left nephrectomy only. Eight weeks after transplantation, all rats underwent right nephrectomy. We measured the life spans of the rats from the time of right nephrectomy (Fig. S8).

Experiment 4.

E30 cloacas dissected from cloned pig fetuses were implanted in the omenta of syngeneic, adult cloned pigs (Fig. S9). Four weeks after implantation, we removed each host animal’s native left kidney and connected the left ureter to the implanted cloaca bladder. One day or four weeks after this surgery (4 or 8 wk after cloacal transplantation), the developed cloacas were removed for examination.

SCNT.

SCNT was conducted as described previously (8, 18), using in vitro-matured oocytes as the recipient cytoplasts. Primary culture cells of porcine fetal fibroblasts (female) were prepared as nuclear donors after cell-cycle synchronization, which was accomplished using serum starvation (FBS, 0.5% vol/vol) for 48 h. A single donor cell was inserted into the perivitelline space of an enucleated oocyte. Membrane fusion of the donor cells and recipient cytoplasts was induced electrically. The reconstructed embryos then were activated electrically, followed by in vitro culture for 1–6 d and subsequent transfer to the reproductive tracts of estrus-synchronized recipient gilts.

Metanephroi and Cloaca Transplantation.

Pregnant sows were killed under general anesthesia. E15 rat metanephroi or cloacas and E30 pig metanephroi or cloacas were dissected from fetuses under stereomicroscope guidance. Metanephroi and cloacas were implanted in the omenta of anesthetized host animals (Fig. S5) as previously reported (10, 11, 19).

Cryopreservation of Embryonic Metanephroi.

Pig metanephroi were cryopreserved using the previously reported vitrification method (8, 20), with slight modifications. Briefly, metanephroi were initially equilibrated with 7.5% (vol/vol) ethylene glycol (EG) and 7.5% (vol/vol) DMSO in handling medium [HM; 20 mM Hepes-buffered tissue culture medium 199 + 20% (vol/vol) calf serum] for 25 min, followed by a second equilibration in vitrification solution (VS), consisting of 15% (vol/vol) EG and 15% (vol/vol) DMSO in HM, for 20–50 min, on ice. Two metanephroi per device were placed on a Cryotop device (Kitazato BioPharma) with a minimum volume of VS and were plunged directly into liquid nitrogen for storage (3–36 mo). For warming, the Cryotop was immersed directly in HM containing 1 M sucrose for 1 min at 38.5 °C, followed by stepwise dilution of the cryoprotectants at room temperature. The metanephroi were transferred into HM with 0.5 M sucrose for 3 min and were washed twice in HM for 5 min before transplantation.

SWPU System.

E15 rat cloacas and E30 porcine cloacas were implanted in the para-aortic or parasplenic artery areas of anesthetized host animals. Four weeks after implantation, the left native kidneys were removed, and the left ureters were connected to the bladders developed from the implanted cloacas (Fig. S1A).

Histochemical Analysis.

For histologic analysis, tissues grown from implanted metanephroi or cloacas were placed into 4% (wt/vol) paraformaldehyde in phosphate buffer. The fixed tissues were embedded in paraffin and cut into 5-μm sections. Masson’s trichrome and H&E staining were performed, as described elsewhere (7). The dimensions of the tubular lumen and the degree of interstitial fibrosis were analyzed quantitatively in 10 high-power fields of the cortical area per section, using MetaValue software (Molecular Devices) to determine the fibrotic areas in the sections stained with Masson’s trichrome (blue) and in the areas of renal tubules showing dilation. The maximal 2D areas of the developed metanephroi were determined from 10 serial sections, before and after the presumed largest section, and the glomerular numbers were determined by calculating the number of glomeruli per total renal cortical area of the maximum section, measured using MetaValue software. For immunohistochemical staining of transitional epithelium cells, goat anti-Uroplakin III polyclonal antibody (sc-15182; Santa Cruz Biotechnology) was used.

Blood and Urine Biochemistry.

Blood and urine samples for biochemical analyses were obtained from the inferior vena cava and bladder, respectively, of isoflurane-anesthetized rats. Serum and urine UN and Cr levels were analyzed according to the manufacturer’s instructions (SRL), as reported previously (7).

I.v. Urography.

To analyze whether urine from a bladder grown from a cloacal transplant could be discharged into a recipient bladder via the recipient ureter, CT scans using an Activion 16 CT system (Toshiba Medical Systems) and Omnipaque contrast medium (Daiichi-Sankyo) were performed after rats were killed.

Statistical Analysis.

Data are presented as means ± SEs of measurement. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software). The significance of the differences between two mean values was determined using an unpaired t test. Multiple comparisons involving more than three groups were performed by ANOVA. Survival curves were created using Kaplan–Meier survival analyses.