Single cell RNA sequencing of kidney tubuloids and the tissue that the tubuloids were derived from
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ABSTRACT: Adult Stem Cell (ASC )-derived organoids are 3D epithelial structures that recapitulate essential aspects of their organ of origin. We have developed conditions for the long-term growth of primary kidney tubular epithelial organoids (‘tubuloids’). Cultures can be established from mouse and human kidney tissue, as well as from urine and can be expanded for at least 20 passages (> 6 months). The structures retain a normal number of chromosomes. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. BK virus infection of tubuloids recapitulates in vivo phenomena. "Tumoroids" can be established from Wilms nephroblastoma. Kidney tubuloids from urine from a subject with Cystic Fibrosis (CF) allows ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function. Adult kidney-derived epithelial tubuloids allow studies of hereditary, infectious and malignant kidney disease in a personalized fashion.
Project description:Adult Stem Cell (ASC )-derived organoids are 3D epithelial structures that recapitulate essential aspects of their organ of origin. We have developed conditions for the long-term growth of primary kidney tubular epithelial organoids (‘tubuloids’). Cultures can be established from mouse and human kidney tissue, as well as from urine and can be expanded for at least 20 passages (> 6 months). The structures retain a normal number of chromosomes. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. BK virus infection of tubuloids recapitulates in vivo phenomena. "Tumoroids" can be established from Wilms nephroblastoma. Kidney tubuloids from urine from a subject with Cystic Fibrosis (CF) allows ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function. Adult kidney-derived epithelial tubuloids allow studies of hereditary, infectious and malignant kidney disease in a personalized fashion.
Project description:Adult Stem Cell (ASC)-derived organoids are 3D epithelial structures that recapitulate essential aspects of their organ of origin. We have developed conditions for the long-term growth of primary kidney epithelial organoids. Cultures can be established from mouse and human kidney tissue, as well as from urine and can be expanded for at least 20 passages (> 6 months). Chromosome numbers remain normal. Human organoids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. BK virus infection of organoids recapitulates in vivo phenomena. Organoids can be established from Wilms nephroblastoma. Kidney organoids from Cystic Fibrosis (CF) patient’s urine allowed ex vivo assessment of treatment efficacy. Finally, organoids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active transport function. Adult kidney-derived organoids allow studies of hereditary, infectious and malignant kidney disease in a personalized fashion.
Project description:Adult kidney organoids have been described as strictly tubular epithelial and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR/Cas9 gene-edited PKD1 and PKD2 knockout tubuloids and human ADPKD and control tissue show similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, shows a significant effect on cyst size in tubuloids but no effect in a pluripotent-stem-cell-derived model. Thus, tubuloids derive from a tubular epithelial subpopulation and represent an advanced model for ADPKD disease modeling.
Project description:The aim of the project was the analysis of the bulk proteome and phosphoproteome in 3D organoids (tubuloids) from the adult mouse kidney. Proteome (nearly 9000 proteins) and phosphoproteome (nearly 16000 phospho sites) were compared in three biological (independent) replicates of epithelial organoids (early passage and long-term, 3-month cultures) and freshly isolated, MAC-sorted, Epcam-positive kidney epithelial cells. After collection of the organoids, Matrigel was removed using a non-enzymatic solution.
Project description:Acute kidney injury (AKI) is a frequent and challenging clinical condition associated with high morbidity and mortality. In AKI, renal tubular epithelial cells (TECs) are a primary site of damage, and recovery from AKI depends on TEC plasticity. However, the molecular mechanisms underlying adaptation and maladaptation of TECs in AKI remain largely unclear. Here, our analyses of mouse kidney tubuloids showed that TEC injury resulted in activation of the glucocortiocoid receptor by endogenous glucocorticoids, which aggravated tubular damage. The detrimental effect of endogenous glucocorticoids on injured TECs was exacerbated by the administration of the widely clinically used synthetic glucocorticoid, dexamethasone, as indicated by experiments in mouse kidney tubuloids. Mechanistically, studies in mouse tubuloids demonstrated that glucocorticoid receptor signaling in injured TECs orchestrated a maladaptive transcriptional program to hinder DNA repair and to amplify injury-induced DNA double-strand break formation, as well as to dampen mTOR activity and mitochondrial bioenergetics. This study identifies glucocortiocoid receptor activation as a mechanism of epithelial maladaptation, which is functionally important for acute kidney injury.
Project description:Acute kidney injury (AKI) is a frequent and challenging clinical condition associated with high morbidity and mortality. In AKI, renal tubular epithelial cells (TECs) are a primary site of damage, and recovery from AKI depends on TEC plasticity. However, the molecular mechanisms underlying adaptation and maladaptation of TECs in AKI remain largely unclear. Here, our analyses of mouse kidney tubuloids showed that TEC injury resulted in activation of the glucocortiocoid receptor by endogenous glucocorticoids, which aggravated tubular damage. The detrimental effect of endogenous glucocorticoids on injured TECs was exacerbated by the administration of the widely clinically used synthetic glucocorticoid, dexamethasone, as indicated by experiments in mouse kidney tubuloids. Mechanistically, studies in mouse tubuloids demonstrated that glucocorticoid receptor signaling in injured TECs orchestrated a maladaptive transcriptional program to hinder DNA repair and to amplify injury-induced DNA double-strand break formation, as well as to dampen mTOR activity and mitochondrial bioenergetics. This study identifies glucocortiocoid receptor activation as a mechanism of epithelial maladaptation, which is functionally important for acute kidney injury.
Project description:Kidney diseases including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health burden. Organ trans-plantation or kidney dialysis are the only effective available therapeutic tools. Therefore, in vitro models of kidney diseases and the development of prospective therapeutic means are highly demanded. Within the kidney, the glomeruli are involved in blood filtration and waste excre-tion, and are easily affected by changing cellular conditions. Puromycin aminonucleoside (PAN) is a nephrotoxin, which can be harnessed for imitating acute glomerular damage and modelling of glomerular disease. For this reason, we generated kidney organoids from three iPSC lines and treated these with PAN in order to induce kidney injury. Morphological observations re-vealed disruption of glomerular and tubular structures within the kidney organoids upon PAN treatment, which were confirmed by transcriptome analyses. Subsequent analyses revealed an upregulation of immune response as well as inflammatory and cell death-related processes. We concluded that treatment of iPSC-derived kidney organoids with PAN induces kidney injury mediated an intertwined network of inflammation, cytoskeletal re-arrangement, DNA damage, apoptosis and cell death. Furthermore, urine stem cell-derived kidney organoids can be used for modelling of kidney-associated diseases and drug discovery.
Project description:Tubuloids are adult stem cell-based kidney organoids with promising application in kidney disease modelling and drug screening. However, tubuloids still present limited transepithelial transport capacity, which is essential for the kidney’s function. The extracellular vesicles (EVs) are cell-derived vesicular structures known to regulate several cellular processes, including development and maturation. This study explores the potential of EVs derived from renal cells to support tubuloid functional maturation by increasing the levels of organic anion transport 1 (OAT1), a protein involved in renal waste handling.
Project description:This study reports the cellular self-organization of primary human renal proximal tubule epithelial cells (RPTECs) around a minimal Matrigel scaffold to produce basal-in and apical-out proximal tubule organoids (tubuloids). These tubuloids are produced and maintained in hanging drop cultures for 90+ days, the longest such culture of any kind reported to date. The tubuloids upregulate maturity markers, such as aquaporin-1 (AQP1) and megalin (LRP2), and exhibit less mesenchymal and proliferation markers, such as vimentin and Ki67, compared to 2D cultures. They also experience changes over time as revealed by a comparison of gene expression patterns of cells in 2D culture and in day 31 and day 67 tubuloids. Gene expression analysis and immunohistochemistry reveal an increase in the expression of megalin, an endocytic receptor that can directly bind and uptake protein or potentially assist protein uptake. The tubuloids, including day 90 tubuloids, uptake fluorescent albumin and reveal punctate fluorescent patterns, suggesting functional endocytic uptake through these receptors. Furthermore, the tubuloids release kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, when exposed to albumin in both dose- and time-dependent manners. While this study focuses on potential applications for modeling proteinuric kidney disease, the tubuloids may have broad utility for studies where apical proximal tubule cell access is required.
Project description:The goal of this study was to analyse the effects of the SFPQ-TFE3 fusion by transforming kidney tubuloids with the construct. Samples without the construct (luciferase only) and overexpressing the partner only (TFE3) were also used as controls. Furthermore, transformed organoids were also transferred into mice PDX models to check their tumorigenic potential; tumors derived from the organoids were subsequently used for sequencing.