(D) Horizontal (still left -panel) and coronal (best -panel) histologic sights of induced podocytes (201B7) at day 9 stained with hematoxylin and eosin (and were quite low compared with those in human adult podocytes, whereas the expression of was maintained to some extent, consistent with a previous report
(D) Horizontal (still left -panel) and coronal (best -panel) histologic sights of induced podocytes (201B7) at day 9 stained with hematoxylin and eosin (and were quite low compared with those in human adult podocytes, whereas the expression of was maintained to some extent, consistent with a previous report.5 In addition, we detected modest expression in undifferentiated hiPSCs, at about 1/1000 the level in human adult podocytes, as reported previously.11,13 Thus, ABT-639 human podocytes and model of PAN-induced injury46 and patients with nephrotic syndrome.47 Thus, our hiPSC-derived podocytes, which show molecular, morphologic, and functional characteristics of podocytes, will serve as a valuable resource for disease modeling, nephrotoxicity testing. Discussion In this study, we have established a highly efficient podocyte induction method from hiPSCs by combining an NPC sorting method and elucidation of the podocyte specification signals. In the kidney developmental biology field, the signals that dictate the differentiation and patterning of nephron segments from NPCs have been a major focus of interest. differentiation process, demonstrating that phase-specific manipulation of Wnt and Tgf-signaling is critical for podocyte differentiation. Using this insight into the nephron-patterning process, they were able to selectively induce human PSC-derived podocytes with molecular, morphologic, and functional characteristics of ABT-639 human podocytes. This novel protocol will facilitate accessibility to human podocytes, and these PSC-derived podocytes are expected to serve as a valuable resource in kidney research. signaling is critical for podocyte differentiation. First, optimal timing and intensity of Wnt signaling were essential for mesenchymal-to-epithelial transition and podocyte differentiation. Then, inhibition of Tgf-signaling supported domination of the RV proximal domain. Inhibition of Tgf-signaling in the third phase enriched the podocyte fraction by suppressing development of other nephron lineages. The resultant protocol enabled successful induction of human podocytes from PSCs with >90% purity. The induced podocytes exhibited global gene expression signatures comparable to those of adult human podocytes, had podocyte morphologic features (including foot processClike and slit diaphragmClike structures), and showed functional responsiveness to drug-induced injury. Conclusions Elucidation of signals that induce podocytes during the nephron-patterning process enabled ABT-639 us to establish a highly efficient method for selective induction of human podocytes from PSCs. These PSC-derived podocytes show molecular, morphologic, and functional characteristics of podocytes, and PROK1 offer a new resource for disease modeling and nephrotoxicity testing. Podocyte disorders manifest as nephrotic syndrome and/or glomerulosclerosis, which progress to renal failure. Thus, growing attention has been paid to ABT-639 podocyte research in recent years.1,2 Owing to the poor availability of primary human podocytes, artificially immortalized podocyte cell lines3,4 have made great contributions to many podocyte studies. However, these cells do not retain the original characteristics of podocytes, including abundant expression of slit diaphragmCassociated genes and proteins.5 The lack of resources for podocytes with sufficient functional characteristics has been a bottleneck in this field. We and others previously developed methods for induction of nephron progenitor cells (NPCs) from pluripotent stem cells (PSCs), enabling derivation of kidney organoids.6C9 Molecular profiling of the sorted podocytes, comprising approximately 7.5% of the human kidney organoids, confirmed characteristic features that were shared with murine and human podocytes.10 Recent progress in the kidney organoid field has achieved higher-order organization.9 However, it remains a challenge to selectively induce podocytes by controlling the nephron-patterning process from NPCs. Although several groups have reported methods for induction of podocyte-like cells from human induced PSCs (hiPSCs),11C13 the resultant cells expressed only a few selected marker genes at quite low levels and lacked typical slit diaphragm formation. We reasoned that this issue could be addressed by sufficient understanding of the podocyte specification process and signaling from NPCs. The kidney develops by interactions of ureteric bud (UB) and metanephric mesenchyme (MM). The MM includes NPCs and stromal progenitors,14 the former of which express transcription factor and give rise to epithelial nephrons.15 Wnt signaling from the UB triggers condensation of a subset of NPCs below the UB tip to form the pretubular aggregate (PA), followed by the epithelial renal vesicle (RV).16,17 These steps are designated mesenchymal-to-epithelial transition (MET). The RV shows proximodistal polarization, at least by gene expression levels.18C20 Each part of the RV further elongates along the proximodistal axis and differentiates into committed nephron segments, including podocytes, parietal epithelial cells (PECs), proximal tubules (PTs), and distal tubules (DTs). Previous genetic studies revealed the requirement and sufficiency of Wnt signaling for the MET process.16,17,21C23 Accordingly, experiments demonstrated the sufficiency of transient Wnt signaling for MET induction in the isolated MM.24,25 Furthermore, a recent study showed both promotional and suppressive roles of Wnt signaling during the later phase (after RV formation) of distal and proximal nephron development, respectively.26 However, the patterning mechanism for the proximodistal domain of the RV as well as the signals that specify the podocyte lineage during the later process ABT-639 of nephron patterning remain to be elucidated. In this study, we investigated the podocyte lineageCspecification factors by dissecting the nephron development process into three distinct steps: NPCs to PA, PA to RV, and RV to podocytes. For this purpose, we initially employed mouse embryonic NPCs, and then applied the findings in the mouse experiments to hiPSC-derived NPCs to establish a method for selective induction of human podocytes. Methods Animals MafB-GFP knock-in (MafB-GFP) mice were described previously.27 Six2-GFP-Cre transgenic (Six2-GFP) mice15 were kindly provided by Dr. Andrew P. McMahon (University of Southern California). All animal experiments were performed in accordance with institutional guidelines and approved by the Licensing Committee of Kumamoto University (A29C040). Podocyte Induction from Mouse NPCs Metanephroi were isolated from embryonic day (E) 15.5 MafB-GFP embryos and manually minced in PBS(?) using forceps. Minced tissues were dissociated by incubation in 0.25% trypsin-EDTA at 37C for 8 minutes. After blocking with normal mouse.