A Toolbox to Characterize Human Induced Pluripotent Stem Cell-Derived Kidney Cell Types and Organoids.

BACKGROUND: The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible. METHODS: We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics. RESULTS: Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments. CONCLUSIONS: We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.

Detection of Atherosclerotic Inflammation by 68Ga-DOTATATE PET Compared to [18F]FDG PET Imaging.

BACKGROUND: Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([18F]FDG PET), [18F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover. OBJECTIVES: This study tested the efficacy of gallium-68-labeled DOTATATE (68Ga-DOTATATE), a somatostatin receptor subtype-2 (SST2)-binding PET tracer, for imaging atherosclerotic inflammation. METHODS: We confirmed 68Ga-DOTATATE binding in macrophages and excised carotid plaques. 68Ga-DOTATATE PET imaging was compared to [18F]FDG PET imaging in 42 patients with atherosclerosis. RESULTS: Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo 68Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). 68Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBRmax) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). 68Ga-DOTATATE mTBRmax predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and [18F]FDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). [18F]FDG mTBRmax differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial [18F]FDG spillover rendered coronary [18F]FDG scans uninterpretable in 27 patients (64%). Coronary 68Ga-DOTATATE PET scans were readable in all patients. CONCLUSIONS: We validated 68Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that 68Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than [18F]FDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography [VISION]; NCT02021188).