Vascular deficiencies in renal organoids and ex vivo kidney organogenesis.
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ABSTRACT:
SUBMITTER: Ryan AR
PROVIDER: S-EPMC8382085 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Screening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregation in vitro assays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect development in vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.
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Project description:Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.
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Project description:BACKGROUND:Cell-based perfusion studies have provided great insight into fluid-sensing mechanisms, such as primary cilia in the renal and vascular systems. However, the intrinsic limitations of in vitro cell culture, such as the inability to reflect cellular organization within tissues, has distanced observed paradigms from possible clinical developments. Here we describe a protocol that applies ex vivo artery perfusion and calcium imaging to observe real-time cellular responses to fluid-shear stress. RESULTS:Through our ex vivo artery perfusion method, we were able to simulate physiological flow and initiate distinct fluid shear stress mechanosensory responses, as well as induced acetylcholine responses in mouse aortic tissue. The observed calcium profiles confirm results found through previous in vitro cell culture experiments. The overall procedure, including dissection, sample preparation and perfusion, takes around 3 hours to complete. CONCLUSION:Through our unique method, we are able to induce laminar flow within intact mouse aortic tissue and illicit subsequent cellular responses. This method of ex vivo artery perfusion provides the opportunity to bridge the novel findings of in vitro studies with subsequent physiological models of fluid-shear stress mechanosensation in vascular tissues.
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Project description:Extracellular histones are cytotoxic molecules involved in experimental acute kidney injury. In patients receiving a renal transplant from donors after circulatory death, who suffer from additional warm ischemia, worse graft outcome is associated with higher machine perfusate extracellular histone H3 concentrations. We now investigated temperature-dependent extracellular histone release in an ex vivo porcine renal perfusion model, and subsequently studied histone release in the absence and presence of non-anticoagulant heparin. Seven pairs of ischemically damaged porcine kidneys were machine perfused at 4°C (cold ischemia) or 28°C (warm ischemia). Perfusate histone H3 concentration was higher after warm as compared to cold ischemia (median (IQR) = 0.48 (0.20-0.83) μg/mL vs. 0.02 (0.00-0.06) μg/mL; p = .045, respectively). Employing immune-electron microscopy (EM), histone containing cytoplasmic protrusions of tubular and endothelial cells were found after warm ischemic injury. Furthermore, abundant histone localization was detected in debris surrounding severely damaged glomerular cells, in a "buck shot" pattern. In vitro, histones were cytotoxic to endothelial and kidney epithelial cells in a temperature-dependent manner. In a separate ex vivo experiment, addition of heparin did not change the total histone H3 levels observed in the perfusate but revealed a continuous increase in the level of a lower molecular weight histone H3 variant. Our findings show that ischemically damaged kidneys release more extracellular histones in warm ischemia, which by EM was due to histone release by renal cells. Blocking of histone-mediated damage during transplantation may be beneficial in prevention of renal injury.
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Project description:Several three-dimensional cell culture systems are currently available to create liver organoids. In gneral, these systems display better physiologic and metabolic aspects of intact liver tissue compared with two-dimensional culture systems. However, none reliably mimic human liver development, including parallel formation of hepatocyte and cholangiocyte anatomical structures. Here, we show that human fetal liver progenitor cells self-assembled inside acellular liver extracellular matrix scaffolds to form three-dimensional liver organoids that recapitulated several aspects of hepatobiliary organogenesis and resulted in concomitant formation of progressively more differentiated hepatocytes and bile duct structures. The duct morphogenesis process was interrupted by inhibiting Notch signaling, in an attempt to create a liver developmental disease model with a similar phenotype to Alagille syndrome. Conclusion: In the current study, we created an in vitro model of human liver development and disease, physiology, and metabolism, supported by liver extracellular matrix substrata; we envision that it will be used in the future to study mechanisms of hepatic and biliary development and for disease modeling and drug screening. (Hepatology 2018;67:750-761).
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Project description:To evaluate the effect of perfusion status on elasticity measurements of different compartments in the kidney using shear wave elastography (SWE) both in vivo and ex vivo.Thirty-two rabbit kidneys were used to observe the elasticity variation caused by renal artery stenosis and vein ligation in vivo, and six beagle kidneys were studied ex vivo to explore the effect of renal perfusion on elasticity. Supersonic SWE was applied to quantify the elasticity values of different renal compartments (cortex, medulla and sinus). Additionally, histopathological examination was performed to explore the possible mechanisms.The elasticity of the cortex was higher than that of the medulla, and the elasticity of the sinus was lowest among the compartments in native kidneys. The Young's modulus (YM) of the cortex, medulla and sinus increased gradually as the duration of renal vein ligation increased, from 16.34 ± 1.01 kPa to 55.06 ± 5.61 kPa, 13.71 ± 1.16 kPa to 39.63 ± 2.91 kPa, and 12.61 ± 0.84 kPa to 29.30 ± 2.04 kPa, respectively. In contrast, the YM of the three compartments respectively decreased with progressive artery stenosis, from 16.34 ± 1.83 kPa to 11.21 ± 1.79 kPa, 13.31 ± 1.67 kPa to 8.07 ± 1.37 kPa, and 12.78 ± 2.66 kPa to 6.72 ± 0.95 kPa. Artery perfusion was the main factor influencing elasticity in ex vivo. The cortical elasticity was more prone to change with renal perfusion both in vivo and ex vivo. Histopathological examination showed progressive changes in the structure and content of the three compartments, consistent with the elasticity variation.Both the complex structure/anisotropy and the perfusion of the kidney obviously influence the evaluation of renal elasticity. The measurement of SWE should be performed at a specific location along a certain angle or direction, and renal perfusion status should also be taken into account to ensure reproducible detection.
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Project description:Kidney transplantation remains the gold standard treatment for patients suffering from end-stage kidney disease. To meet the constantly growing organ demands grafts donated after circulatory death (DCD) or retrieved from extended criteria donors (ECD) are increasingly utilized. Not surprisingly, usage of those organs is challenging due to their susceptibility to ischemia-reperfusion injury, high immunogenicity, and demanding immune regulation after implantation. Lately, a lot of effort has been put into improvement of kidney preservation strategies. After demonstrating a definite advantage over static cold storage in reduction of delayed graft function rates in randomized-controlled clinical trials, hypothermic machine perfusion has already found its place in clinical practice of kidney transplantation. Nevertheless, an active investigation of perfusion variables, such as temperature (normothermic or subnormothermic), oxygen supply and perfusate composition, is already bringing evidence that ex-vivo machine perfusion has a potential not only to maintain kidney viability, but also serve as a platform for organ conditioning, targeted treatment and even improve its quality. Many different therapies, including pharmacological agents, gene therapy, mesenchymal stromal cells, or nanoparticles (NPs), have been successfully delivered directly to the kidney during ex-vivo machine perfusion in experimental models, making a big step toward achievement of two main goals in transplant surgery: minimization of graft ischemia-reperfusion injury and reduction of immunogenicity (or even reaching tolerance). In this comprehensive review current state of evidence regarding ex-vivo kidney machine perfusion and its capacity in kidney graft treatment is presented. Moreover, challenges in application of these novel techniques in clinical practice are discussed.
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Project description:BackgroundThe shortage of donor organs for transplantation remains a worldwide problem. The utilization of suboptimal deceased donors enlarges the pool of potential organs, yet consequently, clinicians face the difficult decision of whether these sub-optimal organs are of sufficient quality for transplantation. Novel technologies could play a pivotal role in making pre-transplant organ assessment more objective and reliable.MethodsEx vivo normothermic machine perfusion (NMP) at temperatures around 35-37°C allows organ quality assessment in a near-physiological environment. Advanced magnetic resonance imaging (MRI) techniques convey unique information about an organ's structural and functional integrity. The concept of applying magnetic resonance imaging during renal normothermic machine perfusion is novel in both renal and radiological research and we have developed the first MRI-compatible NMP setup for human-sized kidneys.ResultsWe were able to obtain a detailed and real-time view of ongoing processes inside renal grafts during ex vivo perfusion. This new technique can visualize structural abnormalities, quantify regional flow distribution, renal metabolism, and local oxygen availability, and track the distribution of ex vivo administered cellular therapy.ConclusionThis platform allows for advanced pre-transplant organ assessment, provides a new realistic tool for studies into renal physiology and metabolism, and may facilitate therapeutic tracing of pharmacological and cellular interventions to an isolated kidney.
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Project description:Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFβ or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFβ and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.
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