Fig 1: Immunofluorescence staining and electron micrograph images of human podocytes derived from iPSCs(A) Immunostaining of derived podocytes showing expression of podocyte lineage characterization markers WT1, MAFB, Synaptopodin, PODX, Podocin, Nephrin, and F actin for cytoskeleton. Scale bar is 25 μm.(B) Scanning electron microscopy images of iPSC podocytes showing cell bodies with cytoplasmic projections extending to adjacent cells (white arrows). Scale bar is 25 μm.(C) TEM images of the iPSC-derived podocytes showing tight junction-like structures between adjacent cell types (black arrows). Scale bar is 400 nm.
Fig 2: Integration of iPSC-derived podocytes into mouse embryonic kidneys. A Brightfield images of the recombinant organoids generated using iPSC-podocytes and E12.5 embryonic kidney at day 2 and day 7 after initiation of ex vivo co-culture. B Higher (40x, i) and lower (20x, ii) magnification images showing the iPSC-derived podocytes (human-specific MAFB+, red) incorporated into mouse glomerular structures (mouse-specific NPHS1+, green) (n = 2). Counterstaining with DAPI (blue). Scale bar is 50 μm
Fig 3: Characterization of podocyte cells derived from iPSCs(A) Lower and higher magnification brightfield images of the day 12 podocytes derived from the DYR0100 iPSC cell line.(B) Flow cytometry analysis of the DYR0100-derived podocytes for podocyte markers MAFB and PODX.(C) Lower and higher magnification brightfield images of the day 12 podocytes derived from MAFB:mTagBFP2/GATA3:mCherry iPSC cell line.(D) Flow cytometry analysis of the derived podocytes for expression of podocyte marker MAFB and the MAFB promoter-driven mTagBFP2 stained with a FITC secondary antibody. The scale bar is 50 μm.
Fig 4: Comparison of the protocol with existing protocols. A Schematic of the podocyte differentiation protocols compared including key media components. B(i) Comparison of the podocyte markers MAFB and PODXL expression using flow cytometry analysis (n = 3). (ii) Quantification of (Bi). C Immunostaining of iPSC-podocytes with markers PODXL, NPHS1 in the compared protocols (n = 3). Scale bar 100 μm. D Western blot analysis of the podocyte markers PODXL, SYNPO, NPHS1, and WT1 as well as loading control β-actin in the compared protocols (n = 3)
Fig 5: Characterization of human podocytes derived from iPSCs in 12 days. A Immunostaining of iPSC-podocytes showing podocyte lineage markers PODXL (green), P-CADHERIN (green), MAFB (red), SYNPO (green), and F-actin (red) for cytoskeleton. Scale bar is 100 μm. B Pseudo-colored scanning electron microscopy image of an iPSC-podocyte in blue showing large cell bodies with foot-like projections and extracellular vesicles. Scale bar is 30 μm. C Transmission electron microscopy image of podocytes showing tight junction-like structures between cells (black arrows). Scale bar is 400 nm. D Flow cytometry histogram plots of the podocyte markers SYNPO and WT1 stained with a FITC and Texas Red secondary antibody (n = 3). E Quantitative analysis of podocyte markers such as PODXL, WT1, SYNPO, and NEPHRIN throughout the differentiation process was analyzed using (i) RT-PCR and (ii) Western blot quantification with β-actin as control. *Indicates p < 0.05
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