Journal: bioRxiv
Article Title: Dynamic Kidney Organoid Microphysiological Analysis Platform
doi: 10.1101/2024.10.27.620552
Figure Lengend Snippet: a , The initial step of the MAP organogenesis, involving CHIR99021 priming of NPCs, emphasizes the crucial role of secondary signaling in addition to canonical Wnt/β-catenin signaling. The dynamic flow within MAP demonstrates that this signaling occurs in the context of secretion and receptor binding, functioning in an autocrine/paracrine manner. Computational and experimental results underscore the importance of secretion molecules, independent of Wnt/β-catenin easily governed by CHIR99201 perfusion, in facilitating the transition of NPCs into early epithelial nephron constructs. b , MAP’s precision in NPC differentiation signaling allows for the biochemical modulation of organoid development into either proximal-enriched or distal-enriched nephron development through the timely application of CHIR99021 and Y-27632. Optical monitoring within MAP exhibits the distinct developmental trajectories of these two types. Both patterns share a similar initial phase in NPC nephrogenesis, while tubules and stromal cells thrive in the later phases of the enhanced distal development. c , Structural analysis using confocal microscopy reveals the characteristic features of the proximal and the distal enrichment, especially significant distinction of distal tubules and stromal cells. The labels PODXL, LTL, ECAD, and DAPI correspond to podocalyxin, lotus tetragonolobus lectin, E-cadherin, and 4’,6-diamidino-2-phenylindole, respectively. d , The structural arrangement of MAP’s organoids with enhanced distal development reflects a sequential placement from PODXL-positive glomerulus-like structures, proximal tubules, and distal tubules with stroma, resembling early-stage kidney arrangement (Data are represented as the mean ± s.d. from 5 organoids in three independent experiments; data points excluded for visual clarity). e , Quantification of individual compartments indicates the prevalence of E-CAD positive distal tubules and the stromal population in enhanced distal development (EDD) compared to enhanced proximal development (EPD) (n EPD =10 and n EDD =5 from two and three independent experiments, respectively, and presented as the mean ± s.d). The normalized expression levels of the proximal tubule, distal tubule, and stroma showed a significance level in the t -test of p < 0.01 (***).
Article Snippet: The antibodies used in this study included anti-podocalyxin (AF1658, goat, R&D Systems, Inc), anti-E-Cadherin (ab11512, rat, Abcam), biotinylated Lotus tetragonolobus lectin (B-1325-2, Vector Laboratories), and anti-KIM-1 (MAB1750, mouse, R&D Systems, Inc), diluted at concentrations of 10µg·mL -1 , 5µg·mL -1 , 5µg·mL -1 , and 10µg·mL -1 , respectively.
Techniques: Binding Assay, Construct, Confocal Microscopy, Expressing