Fig. 2

Primary podocytes can be cultured from kidney organoid glomeruli. a Isolated organoid glomeruli show evidence of podocyte cell migration (OrgPods) displaying thin arborized projections (TAPs) (inset). Inverted image shown to provide maximum contrast, scale bar 100 µm. b TAPS from newly emerged podocytes are composed of F-actin shown by phallodin immunofluorescent staining. Inverted image, scale bar 50 µm. c Immunostaining at 36 h post-plating shows a strong positively stained 3D OrgGlom with a migrating 2D OrgPod population. Left panel 2D images, right panel 3D reconstruction of Z-stack. Scale bars 50 µm. d At 48 h post-plating OrgPods display a flattened, arborized morphology with processes connecting adjacent cells (arrow), scale bar 50 µm. e Immunostaining of ciPods for SYNAPTOPODIN showed expression is absent in undifferentiated cells (ciPod: Un), only becoming evident following 14 days induced differentiation at 37 °C (ciPod: Diff). OrgPods also display strong SYNAPTOPODIN protein expression, aligned with F-actin stress fibres. Scale bars 100 µm. f OrgPods express the neonatal Fc receptor (FcRN) and actively endocytose fluorescein isothiocyanate (FITC)-labelled albumin at 37 °C resulting in FITC-accumulation in endosomes on the cell surface. This is process halted when performed at 4 °C. Scale bars 50 µm. g OrgPods stimulated with insulin (10 mg/ml) for 10 min showed cortical reorganisation of their actin cytoskeleton with GLUT4 translocation from a vesicular to plasma membrane localisation. Scale bars 50 µm. All representative images reflect a minimum of three biological replicates. For immunofluorescence, images are shown in greyscale for single channels, and merged images in colour