Crystalline organic monoliths with bicontinuous porosity.

Organic crystals are a promising class of materials for various optical applications. However, it has been challenging to make macroscopic organic crystals with bicontinuous porosity that are applicable to flow chemistry. In this study, a new class of porous materials, cm-scale crystalline organic monoliths (COMs) with bicontinuous porosity, are synthesized by replicating the porous structure of silica monolith templates. The COMs composed of p-terphenyl can take up more than 30 wt% of an aqueous solution, and the photophysical properties of the p-terphenyl crystals are well maintained in the COMs. The relatively high surface area of the COMs can be exploited for efficient Dexter energy transfer from triplet sensitizers on the pore surface. The resulting triplet excitons in the COMs encounter and annihilate, generating upconverted UV emission. The COMs would open a new avenue toward applications of organic crystals in flow photoreaction systems.

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.

Generation and Transfer of Triplet Electron Spin Polarization at the Solid-Liquid Interface.

The photoexcited triplet state of dyes can generate highly polarized electron spins for sensing and dynamic nuclear polarization. However, while triplets exhibit long spin-lattice relaxation times (T1) on the microsecond scale in solids, the polarization quickly relaxes on the nanosecond scale in solution due to the rotational motion of chromophores. Here, we report that the immobilization of dye molecules on a solid surface allows molecular contact with a liquid while maintaining high polarization and long T1 as in a solid. By adsorbing anionic porphyrins on cationic mesoporous silica gel, porphyrin triplets exhibit high polarization and long T1 at the solid-liquid interface of silica and toluene. Furthermore, porphyrin triplets on the solid surface can exchange spin polarization with TEMPO radicals in solution. This simple and versatile method using the solid-liquid interface will open the door for utilizing the photoinduced triplet spin polarization in solution, which has been mainly limited to the solid-state.

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.

Proton Hyperpolarization Relay from Nanocrystals to Liquid Water.

Dynamic nuclear polarization (DNP) using transient electron spin polarization generated by photoexcitation can improve nuclear magnetic resonance (NMR) sensitivity far beyond the thermal equilibrium limit for analysis in life science and drug discovery. However, DNP of liquid water at room temperature remains an important challenge. Here, we propose a new method called hyperpolarization relay, in which the nonequilibrium polarization of electron spins is transferred to proton spins in the nanocrystals and then to proton spins in bulk water. Molecular nanocrystals doped with a polarizing agent that generates a highly polarized photoexcited triplet are synthesized by a reprecipitation method while controlling the size of the nanocrystals. The triplet-DNP sequence of repeated laser and microwave irradiation enhances the NMR signal of bulk water as well as nanocrystals. The smaller size of the nanocrystals increases the efficiency of polarization transfer from the nanocrystals to water due to the increased surface area. A series of control experiments and simulations based on Solomon equations confirmed the hyperpolarization relay mechanism.

Generation of functional chimeric kidney containing exogenous progenitor-derived stroma and nephron via a conditional empty niche.

Generation of new kidneys can be useful in various research fields, including organ transplantation. However, generating renal stroma, an important component tissue for structural support, endocrine function, and kidney development, remains difficult. Organ generation using an animal developmental niche can provide an appropriate in vivo environment for renal stroma differentiation. Here, we generate rat renal stroma with endocrine capacity by removing mouse stromal progenitor cells (SPCs) from the host developmental niche and transplanting rat SPCs. Furthermore, we develop a method to replace both nephron progenitor cells (NPCs) and SPCs, called the interspecies dual replacement of the progenitor (i-DROP) system, and successfully generate functional chimeric kidneys containing rat nephrons and stroma. This method can generate renal tissue from progenitors and reduce xenotransplant rejection. Moreover, it is a safe method, as donor cells do not stray into nontarget organs, thus accelerating research on stem cells, chimeras, and xenotransplantation.

Beneficial Impact of Interspecies Chimeric Renal Organoids Against a Xenogeneic Immune Response.

BACKGROUND: Animal fetal kidneys have the potential to be used as scaffolds for organ regeneration. We generated interspecies chimeric renal organoids by adding heterologous rat renal progenitor cells to single cells from mouse fetal kidneys and applying the renal development mechanism of mouse fetuses to rat renal progenitor cells to examine whether rat renal progenitor cells can differentiate into renal tissues of the three progenitor cell lineages of kidneys between different species. Furthermore, we investigated whether chimeric renal organoids with an increased proportion of recipient cells reduce xenogeneic rejection. METHODS: C57BL/6JJmsSlc mice (B6 mice) and Sprague-Dawley-Tg (CAG-EGFP) rat (GFP rats) fetuses were used as donors, and mature male NOD/Shi-scid, IL-2RγKO Jic mice (NOG mice) and Sprague-Dawley rats (SD rats) were used as recipients. First, fetal kidneys were removed from E13.5 B6 mice or E15.5 GFP rats and enzymatically dissociated into single cells. These cells were then mixed in equal proportions to produce chimeric renal organoids in vitro. The chimeric organoids were transplanted under the renal capsule of NOG mice, and maturation of the renal tissues in the organoids was observed histologically. Furthermore, chimeric organoids were prepared by changing the ratio of cells derived from mouse and rat fetal kidneys and transplanted under the renal capsule of SD rats subjected to mild immunosuppression to pathologically analyze the strength of the xenogeneic immune response. RESULTS: The cap mesenchyme was reconstructed in vitro, and nephron progenitor cells and ureteric buds were mosaically comprised GFP-negative mouse and GFP-positive rat cells. In the in vivo environment of immunodeficient mice, chimeric renal organoids mosaically differentiated and matured into renal tissues of three lineages. Chimeric renal organoids with high rates of recipient rat cells showed milder rejection than complete xenograft organoids. The vessels of recipient rats entered from the periphery of the transplanted chimeric renal organoids, which might reduce their immunogenicity. CONCLUSION: Interspecies chimeric renal organoids may differentiate into mature renal tissues of each renal progenitor cell lineage. Furthermore, they may reduce transplant rejection compared with xenograft organoids.

Triplet Dynamic Nuclear Polarization of Guest Molecules through Induced Fit in a Flexible Metal-Organic Framework.

Dynamic nuclear polarization utilizing photoexcited triplet electrons (triplet-DNP) has great potential for room-temperature hyperpolarization of nuclear spins. However, the polarization transfer to molecules of interest remains a challenge due to the fast spin relaxation and weak interaction with target molecules at room temperature in conventional host materials. Here, we demonstrate the first example of DNP of guest molecules in a porous material at around room temperature by utilizing the induced-fit-type structural transformation of a crystalline yet flexible metal-organic framework (MOF). In contrast to the usual hosts, 1 H spin-lattice relaxation time becomes longer by accommodating a pharmaceutical model target 5-fluorouracil as the flexible MOF changes its structure upon guest accommodation to maximize the host-guest interactions. Combined with triplet-DNP and cross-polarization (CP), this system realizes an enhanced 19 F NMR signal of guest target molecules.

Techniques of fragile renal organoids transplantation in mice.

PURPOSE: This study aimed to develop a microsurgical technique to transplant extremely fragile renal organoids in vivo, created by in-vitro reaggregation of metanephros from fetal mice. These organoids in reaggregation and development were examined histologically after transplantation under the renal capsule. METHODS: Initially, metanephros from fetal mice were enzymatically treated to form single cells, and spheroids were generated in vitro. Under a microscope, the renal capsule was detached to avoid bleeding, and the outer cylinder of the indwelling needle was inserted to detach the renal parenchyma from the renal capsule using water pressure. The reaggregated spheroid was aspirated from the culture plate using a syringe with an indwelling needle outer cylinder and carefully extruded under the capsule. Pathological analysis was performed to evaluate changes in reaggregated spheroids over time and the effects of co-culture of spinal cord and subcapsular implantation on maturation. RESULTS: In vitro, the formation of luminal structures became clearer on day 5. These fragile organoids were successfully implanted without tissue crapes under the renal capsule and formed glomerular. The effect of spinal cord co-transplant was not obvious histrionically. CONCLUSIONS: A simple and easy method to transplant fragile spheroids and renal under the renal capsule without damage was developed.

Techniques of orthotopic renal transplantation. II. Size-matched porcine grafts in monkey recipients.

PURPOSE: As a classical xenotransplantation model, porcine kidneys have been transplanted into the lower abdomen of non-human primates. However, we have improved upon this model by using size-matched grafting in the orthotopic position. The beneficial aspects and surgical details of our method are reported herein. METHODS: Donors were two newborn pigs (weighting 5 to 6 kg) and recipients were two cynomolgus monkeys (weighting, approximately, 7 kg). After bilateral nephrectomy, kidneys were cold-transported in Euro-Collins solution. The porcine kidney was transplanted to the site of a left nephrectomy and fixed to the peritoneum. RESULTS: Kidneys transplanted to the lower abdomen by the conventional method were more susceptible to torsion of the renal vein (two cases). In contrast, early-stage blood flow insufficiency did not occur in orthotopic transplants of theleft kidney. CONCLUSIONS: Size-matched porcine-primate renal grafting using our method of transplanting tothe natural position of the kidneys contributes to stable post-transplant blood flow to the kidney.

TAFRO Syndrome With Kidney Involvement: A Case Series of Patients With Kidney Biopsies.

TAFRO (thrombocytopenia, anasarca, fever, reticulin myelofibrosis/renal insufficiency, and organomegaly) syndrome is a systemic inflammatory disease sharing some features with Castleman disease and POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes) syndrome in relation to abnormal secretions of interleukin 6 and vascular endothelial growth factor. The kidney is a main target organ of TAFRO syndrome but the kidney histopathology associated with TAFRO syndrome is yet to be completely defined. We report 3 TAFRO syndrome cases with different clinical courses in which kidney biopsies were performed. In all 3 cases, kidney biopsies showed similar glomerular lesions of diffuse global swelling of the endothelium and expansion of subendothelial spaces, consistent with severe glomerular endothelial injury. Case 3 showed an additional finding of focal tubulointerstitial injury characterized by marked plasma cell infiltration, which was absent in the other 2 cases. Clinical symptoms in cases 1 and 2, which had lower disease severity scores of TAFRO syndrome, were effectively treated with the administration of corticosteroids or a combination of corticosteroids and cyclosporine A. Case 3, with a higher disease severity score, had an aggressive clinical course that was refractory to corticosteroids and tocilizumab; the patient ultimately died of multiple organ failure. In all 3 cases, kidney biopsy provided indications for the diagnosis process and clinical management of TAFRO syndrome.

In vivo regeneration of neo-nephrons in rodents by renal progenitor cell transplantation.

Renal progenitor cells induced from pluripotent stem cells have attracted attention as a cell source for organ regeneration. Here, we report an in vivo protocol for the regeneration of urine-producing nephrons, i.e., neo-nephrons, in mice. We outline steps to transplant exogenous renal progenitor cells into the nephrogenic zone of transgenic mice and subsequently analyze these neo-nephrons. For complete details on the use and execution of this protocol, please refer to Fujimoto et al. (2020).

Techniques of orthotopic renal transplantation. II. Size-matched porcine grafts in monkey recipients

ABSTRACT Purpose As a classical xenotransplantation model, porcine kidneys have been transplanted into the lower abdomen of non-human primates. However, we have improved upon this model by using size-matched grafting in the orthotopic position. The beneficial aspects and surgical details of our method are reported herein. Methods Donors were two newborn pigs (weighting 5 to 6 kg) and recipients were two cynomolgus monkeys (weighting, approximately, 7 kg). After bilateral nephrectomy, kidneys were cold-transported in Euro-Collins solution. The porcine kidney was transplanted to the site of a left nephrectomy and fixed to the peritoneum. Results Kidneys transplanted to the lower abdomen by the conventional method were more susceptible to torsion of the renal vein (two cases). In contrast, early-stage blood flow insufficiency did not occur in orthotopic transplants of theleft kidney. Conclusions Size-matched porcine-primate renal grafting using our method of transplanting tothe natural position of the kidneys contributes to stable post-transplant blood flow to the kidney.

Techniques of fragile renal organoids transplantation in mice

Purpose: This study aimed to develop a microsurgical technique to transplant extremely fragile renal organoids in vivo, created by in-vitro reaggregation of metanephros from fetal mice. These organoids in reaggregation and development were examined histologically after transplantation under the renal capsule. Methods: Initially, metanephros from fetal mice were enzymatically treated to form single cells, and spheroids were generated in vitro. Under a microscope, the renal capsule was detached to avoid bleeding, and the outer cylinder of the indwelling needle was inserted to detach the renal parenchyma from the renal capsule using water pressure. The reaggregated spheroid was aspirated from the culture plate using a syringe with an indwelling needle outer cylinder and carefully extruded under the capsule. Pathological analysis was performed to evaluate changes in reaggregated spheroids over time and the effects of co-culture of spinal cord and subcapsular implantation on maturation. Results: In vitro, the formation of luminal structures became clearer on day 5. These fragile organoids were successfully implanted without tissue crapes under the renal capsule and formed glomerular. The effect of spinal cord co-transplant was not obvious histrionically. Conclusions: A simple and easy method to transplant fragile spheroids and renal under the renal capsule without damage was developed.

Kidney Transplant Graftectomy by Severe Mixed-Type Rejection with Acute and Chronic Active Vascular Lesions at Entire Levels of the Renal Vasculature.

Vascular lesions related to allograft rejection have a big impact on graft survival. As such, investigation of these lesions is important to understand the pathophysiology of rejection and its management. We report a case of kidney transplant graftectomy by severe mixed-type rejection with acute and chronic active vascular lesions caused by non-adherence to immunosuppressive treatment. The patient presented is a 29-year-old male who received a kidney transplantation in July 2011 (ABO compatible) from his father. He then did not come to the hospital for 3 months prior to his admission and also made his own decision to stop his medication regimen. On October 2013, the patient came to the hospital with dyspnea, nausea, and vomiting and had significant renal dysfunction (serum Cr 30.4 mg/dL, BUN 191 mg/dL). A kidney graft biopsy showed cortical necrosis with severe interstitial hemorrhage and thrombotic microangiopathy (TMA). Despite steroid pulse therapy, kidney graft function did not recover, and the patient underwent a subsequent graft resection. The resected kidney graft displayed various vascular lesions from the renal artery to the interlobular arteries and arterioles including endarteritis, TMA, fibrinoid necrosis, and transplant arteriopathy. This case shows the detailed pathological findings of the vascular lesions in the entire artery tree of kidney allograft, and the pathophysiology is discussed.

Generation of Human Renal Vesicles in Mouse Organ Niche Using Nephron Progenitor Cell Replacement System.

Animal fetuses may be used for the regeneration of human organs. We have previously generated a transgenic mouse model that allows diphtheria toxin (DT)-induced ablation of Six2-positive nephron progenitor cells (NPCs). Elimination of existing native host NPCs enables their replacement with donor NPCs, which can generate neo-nephrons. However, this system cannot be applied to human NPCs, because DT induces apoptosis in human cells. Therefore, the present study presents a transgenic mouse model for the ablation of NPCs using tamoxifen, which does not affect human cells. Using this system, we successfully regenerate interspecies neo-nephrons, which exhibit urine-producing abilities, from transplanted rat NPCs in a mouse host. Transplantation of human induced pluripotent stem cell (iPSC)-derived NPCs results in differentiation into renal vesicles, which connect to the ureteric bud of the host. Thus, we demonstrate the possibility of the regeneration of human kidneys derived from human iPSC-derived NPCs via NPC replacement.