Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A Schematic procedures for kidney organoid differentiation from human PSC lines in static and STR culture systems. The schema explains the maintenance and differentiation process starting from PSCs to nephron progenitor cells (NPCs) and ultimately kidney organoids. STR and static organoids were treated with Activin for 2 or 3 days after 4 days of CHIR ± Dorsomorphin treatment. Spheroids were then treated with both CHIR and FGF9 starting on day 7 or 8 for 2 days, and subsequently, CHIR was removed. From day 13 or 14, no growth factors were added. The right-sided photo compares the final volumes of static (from 6 wells of one plate) and STR organoids, respectively. The scale bars: 25 mm. B Brightfield image of differentiating H9 cells in static and STR systems. The scale bar represents 500 µm. C Confocal images for the expression of nephron progenitor markers (SIX2 and SALL1) by H9 NPCs on day 8 of differentiation in static and STR systems. Scale bars: 100 µm. D Confocal images of day 21 STR-kidney organoids composed of proximal tubules (LTL: Lotus tetragonolobus lectin + ), interstitial cells (MEIS1/2/3 + ), endothelial cells (CD31 + ), podocytes (PODXL: Podocalyxin + ), loops of Henle/distal nephrons (CDH1: E-cadherin + ) with a scale bar of 200 µm. E Comparison of the total cost of goods/materials (COGs; cell culture reagents, media, and materials) in static and STR systems to generate the same cell number of kidney organoids. F Organoid cell numbers per media volume generated from one 96-well plate (static) and one 5-ml bioreactor (STR).

Article Snippet: Collagen IV α1 , Rockland , 600-401-106S, RRID: AB_11183330.

Techniques: Expressing, Comparison, Cell Culture, Generated

Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A-D Principal component ( A ) and hierarchical clustering ( B ) analyses from RNA-seq samples on day 0, day 8 (NPC), static day 21, 35, 49, 63 (Org_21, Org_35, Org_49, Org_63), and STR day 21, 35, 49 (STR_21, STR_35, STR_49). n = 3 mRNA samples. Each mRNA sample was collected from 6-12 organoids. Gene ontology (GO) analysis of DEGs significantly upregulated in STR organoids compared to static ones on day 21 ( C ) and day 35 ( D ). E Live-cell imaging in STR and static organoids to assess the transepithelial cation transport using Rhodamine 123 (Rh123). On days 32–38 of differentiation, both STR and static ones were transferred to the 1% Geltrex-coated 8-well glass-bottom chamber slides. On days 35–39, proximal tubules of organoids in these slides were labeled with LTL conjugated with Alexa Fluor 647. On days 36–40, after the organoids in chamber slides were assigned and mapped by the confocal microscope, the cation Rh123 (10 mM) was added into the culture media at a final concentration of 10 μM. Live-cell imaging was immediately started to check the efficiency of cation transport through LTL+ proximal tubular epithelial cells. Baseline brightfield images show the particular LTL+ proximal tubules before the addition of Rh123. Following the supplementation of Rh123, its uptake by tubular epithelial cells was recorded for 20–30 min via real-time imaging. The images encircled by white dashed lines exhibit the course of Rh123 uptake by LTL+ tubular epithelial cells at 0, 10, and 20 min. F Quantification of apical and basal Rh123 fluorescence intensities of proximal tubular epithelial cells to calculate the apicobasal ratio of Rh123 uptake for the assessment of cation transport from basal to apical sides in these tubular epithelial cells. Each dot represents the proximal tubular histogram analysis of each organoid, n = 6 organoids from 2 independent organoid batches of BJFF.6 and HUES62 cell lines. The asterisk in the bar graph indicates the p value derived from a two-tailed unpaired t-test. Means ± SEM. ** p ≤ 0.01.

Article Snippet: Collagen IV α1 , Rockland , 600-401-106S, RRID: AB_11183330.

Techniques: RNA Sequencing, Live Cell Imaging, Labeling, Microscopy, Concentration Assay, Imaging, Fluorescence, Derivative Assay, Two Tailed Test

Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A Vessel sprouts in polarized podocyte clusters with CD146+ capillary network supported by MEIS1/2/3+ stromal cells in day 21 STR organoids (left set of two images) compared to the podocyte clusters of day 21 control static organoids (right set of four images). White arrows in the STR images indicate the vascularized glomeruli surrounded by polarized podocytes. Scale bars of STR images represent 100 μm and 10 μm (zoomed STR image), respectively. Rectangular white dashed lines point out the zoomed images of non-vascularized podocyte clusters of the static organoid. Scale bars of static organoid images represent 100 μm. B 3D surface reconstruction of CD146+ capillary network and surrounding PODXL+ podocyte clusters as vascularized glomerular components. Z-stack images were acquired at a step size of 2 μm. The scale bar represents 30 μm. C Transmission electron microscopy images show multiple vascular lumens (left image) and accompanying podocyte foot processes around the vascular lumen (right image) within organoid glomerular structures on day 35. The asterisk indicates the nucleus of the podocyte surrounding the vascular lumen, while the arrowheads mark podocyte foot processes. Black scale bars at the corners: 2 μm. D Percentages of vascularized podocyte clusters containing CD146 endothelial surface in STR versus static organoids via 3D surface reconstruction by machine learning. n = 3 static and n = 9 STR organoids from 3 independent batches differentiated from BJFF.6 or HUES62 cells. E ACTA2+ mesangial-like cells surrounded by polarized PODXL+ podocytes in Bowman’s space of day 35 STR and static organoids. STR organoids were generated by the transfer of the same-batch static organoids into STR wells on day 14 of differentiation. The graph represents the percentage of organoids containing ACTA2+ cells ( n = 5 static and n = 6 STR organoids from 2 independent batches differentiated from H9 or HUES62 cells). F Upregulation of certain GO terms associated with extracellular matrix organization, cell adhesion, and migration in day 35 STR organoids compared to static organoids. “Regulation of cell migration”, “Positive regulation of cell adhesion”, “Regulation of cell adhesion”, and “Extracellular matrix organization” terms constitute GO biological processes while “Extracellular matrix”, “Collagen-containing extracellular matrix”, “Actin-based cell projection”, and “Basement membrane”, “Collagen network”, and “Basement membrane collagen trimer” terms can be classified as GO cellular components, and “Assembly of collagen fibrils and other multimeric structures” is a part of Reactome pathways. G Inhibition of glomerular vascularization of STR organoids by the application of α2β1 integrin inhibitor, BTT3033 (stock concentration of 10 mM), with a final concentration of 1 μM for 7 or 18 days, in contrast to the control vehicle-treated organoids. The white arrows in the confocal microscopy images indicate the vascularized glomeruli surrounded by polarized podocytes. The scale bars represent 100 μm. H 3D surface reconstruction of CD146+ capillary network and surrounding PODXL+ podocyte clusters as vascularized glomerular surfaces in vehicle- and BTT3033-treated organoids. Z-stack images were acquired at a step size of 2 μm. The scale bar represents 50 μm. Y-axes of graphs represent two parameters: the percentage of vascularized podocyte clusters and the volume percent ratio of CD146+ vascular surface in these podocyte clusters to the whole PODXL+ podocyte surface, respectively ( n = 7). Asterisks in the bar graphs ( D , E , H ) indicate p values derived from two-tailed unpaired t-tests. Means ± SEM. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: Collagen IV α1 , Rockland , 600-401-106S, RRID: AB_11183330.

Techniques: Control, Transmission Assay, Electron Microscopy, Generated, Migration, Membrane, Inhibition, Concentration Assay, Confocal Microscopy, Derivative Assay, Two Tailed Test

Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A Workflow for the implantation of nephron sheets generated from STR organoids into dorsal skinfold chambers (DSFCs) of NSG mice. Implantation of STR organoids into mice was performed six times from three independent organoid batches. B Phase 1 left photo shows the self-assembled nephron sheet formed by STR organoids. Instead of implantation, this sheet was fixed with 4% PFA on day 28 to confirm the self-assembly process. The right image represents the stained nephron sheet with the designated markers (LTL, CD31, CDH1, CD146, PODXL, MEIS1/2/3) following the fixation process. The scale bars represent 1 mm. C Phase 2 photos include the NSG mouse with a dorsal skin fold chamber (DSFC) on its back (left photo) and the implanted nephron sheet generated from day 24 STR organoids inside the DSFC (right photo). D Phase 3 multiphoton intravital microscopy (MP-IVM) of the implanted sheet reveals the nephron units (designated by a circular white dashed line) with the dynamic flow of both high-molecular-weight (HMW, 500 kDa-Cy5) and low-molecular-weight (LMW, 3 kDa-FITC) dextrans in glomerular vascular-like structures (designated by 3D-reconstructed gray surface), accompanied by the filtration of LMW dextran into the surrounding space. The 3D-reconstructed cellular surface was generated via 3D surface rendering of Hoechst in Z-stack images by IMARIS. The scale bar displays 20 μm. E Phase 4 left photo shows the gross vascularization of the implanted nephron sheet with murine vessels on the 6th day of implantation, following its extraction and fixation. 3D reconstructed image (right) demonstrates the ongoing CD146+ human vascular network inside the same phase 4 nephron sheet (PODXL, CD146, LTL, MEIS123). Z-stack images were acquired at a step size of 100 μm. The scale bars represent 1 mm. F The confocal microscopy image showing the anastomosis of MECA-32-CD146+ human (white arrows, single-positive red) and MECA-32+ murine vascular endothelial networks. The scale bar displays 150 μm.

Article Snippet: Collagen IV α1 , Rockland , 600-401-106S, RRID: AB_11183330.

Techniques: Generated, Staining, Intravital Microscopy, High Molecular Weight, Molecular Weight, Filtration, Extraction, Confocal Microscopy