Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.

Maturation and development of fetal pig intestinal tissue in immunodeficient mice.

PURPOSE: This study aimed to compare the degree of maturation and development of fetal pig segmental intestinal tissue with that of spheroids created by in-vitro reaggregation of dissociated fetal intestinal cells after transplantation into immunodeficient mice. METHODS: Fetal pig small intestines were transplanted as segmental grafts into the omentum and subrenal capsules of immunodeficient mice or enzymatically treated to generate single cells. Spheroids made by in-vitro reaggregation of these cells were transplanted into the subrenal capsules of immunodeficient mice. The segmental grafts and spheroids were harvested four and eight weeks after transplantation, and the structural maturity and in-vivo development of these specimens were histologically evaluated. RESULTS: The spheroids were engrafted and supplied blood vessels from the host mice, but an intestinal layered structure was not clearly observed, and there was almost no change in size. On the other hand, the segmental grafts formed deep crypts in the mucus membrane, the inner circular layer, and outer longitudinal muscles. The crypts of the transplanted grafts harvested at eight weeks were much deeper, and the smooth muscle layer and the enteric nervous system were more mature than those of grafts harvested at the fourth week, although the intestinal peristaltic wave was not observed. CONCLUSIONS: Spheroids created from fetal small intestinal cells could not form layered structures or mature sufficiently. Conversely, segmental tissues structurally matured and developed after in-vivo transplantation and are therefore potential grafts for transplantation.

Exploration of Preservation Methods for Utilizing Porcine Fetal-Organ-Derived Cells in Regenerative Medicine Research.

Human pluripotent stem cells have been employed in generating organoids, yet their immaturity compared to fetal organs and the limited induction of all constituent cell types remain challenges. Porcine fetal progenitor cells have emerged as promising candidates for co-culturing with human progenitor cells in regeneration and xenotransplantation research. This study focused on identifying proper preservation methods for porcine fetal kidneys, hearts, and livers, aiming to optimize their potential as cell sources. Extracted from fetal microminiature pigs, these organs were dissociated before and after cryopreservation-thawing, with subsequent cell quality evaluations. Kidney cells, dissociated and aggregated after vitrification in a whole-organ form, were successfully differentiated into glomeruli and tubules in vivo. In contrast, freezing hearts and livers before dissociation yielded suboptimal results. Heart cells, frozen after dissociation, exhibited pulsating heart muscle cells similar to non-frozen hearts. As for liver cells, we developed a direct tissue perfusion technique and successfully obtained highly viable liver parenchymal cells. Freezing dissociated liver cells, although inferior to their non-frozen counterparts, maintained the ability for colony formation. The findings of this study provide valuable insights into suitable preservation methods for porcine fetal cells from kidneys, hearts, and livers, contributing to the advancement of regeneration and xenotransplantation research.

Maturation and development of fetal pig intestinal tissue in immunodeficient mice

Purpose: This study aimed to compare the degree of maturation and development of fetal pig segmental intestinal tissue with that of spheroids created by in-vitro reaggregation of dissociated fetal intestinal cells after transplantation into immunodeficient mice. Methods: Fetal pig small intestines were transplanted as segmental grafts into the omentum and subrenal capsules of immunodeficient mice or enzymatically treated to generate single cells. Spheroids made by in-vitro reaggregation of these cells were transplanted into the subrenal capsules of immunodeficient mice. The segmental grafts and spheroids were harvested four and eight weeks after transplantation, and the structural maturity and in-vivo development of these specimens were histologically evaluated. Results: The spheroids were engrafted and supplied blood vessels from the host mice, but an intestinal layered structure was not clearly observed, and there was almost no change in size. On the other hand, the segmental grafts formed deep crypts in the mucus membrane, the inner circular layer, and outer longitudinal muscles. The crypts of the transplanted grafts harvested at eight weeks were much deeper, and the smooth muscle layer and the enteric nervous system were more mature than those of grafts harvested at the fourth week, although the intestinal peristaltic wave was not observed. Conclusions: Spheroids created from fetal small intestinal cells could not form layered structures or mature sufficiently. Conversely, segmental tissues structurally matured and developed after in-vivo transplantation and are therefore potential grafts for transplantation.

Long-term viable chimeric nephrons generated from progenitor cells are a reliable model in cisplatin-induced toxicity.

Kidney organoids have shown promise as evaluation tools, but their in vitro maturity remains limited. Transplantation into adult mice has aided in maturation; however, their lack of urinary tract connection limits long-term viability. Thus, long-term viable generated nephrons have not been demonstrated. In this study, we present an approachable method in which mouse and rat renal progenitor cells are injected into the developing kidneys of neonatal mice, resulting in the generation of chimeric nephrons integrated with the host urinary tracts. These chimeric nephrons exhibit similar maturation to the host nephrons, long-term viability with excretion and reabsorption functions, and cisplatin-induced renal injury in both acute and chronic phases, as confirmed by single-cell RNA-sequencing. Additionally, induced human nephron progenitor cells differentiate into nephrons within the neonatal kidneys. Collectively, neonatal injection represents a promising approach for in vivo nephron generation, with potential applications in kidney regeneration, drug screening, and pathological analysis.

Evaluation of the ability of human induced nephron progenitor cells to form chimeric renal organoids using mouse embryonic renal progenitor cells.

The number of patients with end-stage renal failure is increasing annually worldwide and the problem is compounded by a shortage of renal transplantation donors. In our previous research, we have shown that transplantation of renal progenitor cells into the nephrogenic region of heterologous fetuses can induce the development of nephrons. We have also developed transgenic mice in which specific renal progenitor cells can be removed by drugs. By combining these two technologies, we have succeeded in generating human-mouse chimeric kidneys in fetal mice. We hope to apply these technologies to regenerative medicine. The quality of nephron progenitor cells (NPCs) derived from human pluripotent stem cells is important for the generation of chimeric kidneys, but there is currently no simple evaluation system for the chimerogenic potential of human NPCs. In this study, we focused on the fact that the re-aggregation of mouse renal progenitor cells can be used for nephron formation, even when merged into single cells. First, we examined the conditions under which nephron formation is likely to occur in mice during re-aggregation. Next, to improve the differentiation potential of human NPCs derived from pluripotent stem cells, NPCs were sorted using Integrin subunit alpha 8 (ITGA8). Finally, we demonstrated chimera formation between different species by mixing mouse cells with purified, selectively-induced human NPCs under optimum conditions. We observed these chimeric organoids at different time points to learn about these human-mouse chimeric structures at various stages of renal development. We found that the rate of chimera formation was affected by the purity of the human NPCs and the cell ratios used. We demonstrated that chimeric nephrons can be generated using a simple model, even between distant species. We believe that this admixture of human and mouse renal progenitor cells is a promising technology with potential application for the evaluation of the chimera formation abilities of NPCs.

Cryopreservation of Fetal Porcine Kidneys for Xenogeneic Regenerative Medicine.

Kidney xenotransplantation has been attracting attention as a treatment option for end-stage renal disease. Fetal porcine kidneys are particularly promising grafts because they can reduce rejection through vascularization from host vessels. We are proposing xenogeneic regenerative medicine using fetal porcine kidneys injected with human nephron progenitor cells. For clinical application, it is desirable to establish reliable methods for the preservation and quality assessment of grafts. We evaluated the differentiation potency of vitrified porcine fetal kidneys compared with nonfrozen kidneys, using an in vivo differentiation model. Fetal porcine kidneys connected to the bladder were frozen via vitrification and stored in liquid nitrogen. Several days later, they were thawed and transplanted under the retroperitoneum of immunocompromised mice. After 14 days, the frozen kidneys grew and differentiated into mature nephrons, and the findings were comparable to those of nonfrozen kidneys. In conclusion, we demonstrated that the differentiation potency of vitrified fetal porcine kidneys could be evaluated using this model, thereby providing a practical protocol to assess the quality of individual lots.

Acute tubulointerstitial nephritis associated with antineutrophil cytoplasmic antibody following cimetidine treatment: a case report.

BACKGROUND: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis usually induces rapidly progressive glomerulonephritis, including pauci-immune necrotizing crescentic glomerulonephritis. Acute tubulointerstitial nephritis (ATIN), which is often drug-induced, is a frequent cause of kidney injury. However, ATIN associated with ANCA without any glomerular lesions has been rarely reported, and drug-induced ATIN associated with ANCA is not well recognized. Here we present a case of an older woman with ATIN associated with myeloperoxidase-ANCA (MPO-ANCA) following cimetidine treatment. CASE PRESENTATION: A 70-year-old woman was admitted to our hospital due to acute kidney injury and mild proteinuria. She had a one-year history of chronic thyroiditis and dyslipidemia, for which she was taking levothyroxine sodium and atorvastatin, respectively. Two weeks before admission she had started cimetidine, methylmethionine sulfonium chloride, and itopride hydrochloride for gastric discomfort persistent since a month. She had experienced fatigue for two weeks and later appetite loss. The patient demonstrated a positive titer for MPO-ANCA (192 IU/mL) and a positive drug-induced lymphocyte stimulation test for cimetidine. She underwent two kidney biopsies that revealed ATIN without any glomerular lesions. Despite discontinuation of cimetidine on admission, renal injury continued with the presence of high MPO-ANCA titer. Oral steroid treatment was closely related with the recovery of her renal function and disappearance of MPO-ANCA. CONCLUSIONS: In this case, ATIN presented as sustained renal insufficiency and high MPO-ANCA titer despite withdrawal of cimetidine. Therefore, we reason that the development of ANCA-associated ATIN was caused by cimetidine. Serologic follow-up with measurement of MPO-ANCA titers and renal biopsy are recommended when the clinical history is inconsistent with the relatively benign course of drug-induced ATIN.

Maternally inherited diabetes and deafness complicated by mesangial galactose-deficient IgA1 deposits: a case report.

BACKGROUND: Maternally inherited diabetes and deafness (MIDD), a mitochondrial genetic disorder, typically affects the kidneys and results in end-stage renal disease. Early diagnosis of MIDD is challenging when renal manifestations precede other key clinical features such as diabetes and deafness and/or when the disease is complicated by other renal pathologies. CASE PRESENTATION: Here, we present the case of a 33-year-old Japanese woman who had initially been diagnosed with IgA nephropathy but was found to have MIDD 6 years later. Two renal biopsies were conducted six years apart. While assessment of the first biopsy specimen with the monoclonal antibody (KM55) revealed mesangial IgA deposits (containing the galactose-deficient IgA1 variant [Gd-IgA1]), examination of the second specimen showed no mesangial IgA deposits and newly-developed glomerular global scleroses and tubular damage. Granular swollen epithelial cells (GSECs), characterised by abnormal mitochondria, were observed among the tubules and collecting ducts in both biopsy specimens. Mitochondrial DNA analysis revealed an m.3243A > G mutation. CONCLUSIONS: We rediscovered the usefulness of GSECs as a pathologically distinctive feature of mitochondrial nephropathy and reviewed the literature regarding MIDD complicated by mesangial IgA deposition. Furthermore, we demonstrate that the mesangial IgA deposits in this patient consisted of the galactose-deficient IgA1 variant. The monoclonal antibody (KM55) might be a useful tool to distinguish IgAN from latent IgA deposits.