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Manipulation of Nephron-Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells

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Manipulation of Nephron-Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells BASIC RESEARCH www.jasn.org Manipulation of Nephron-Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells Yasuhiro Yoshimura,1,2 Atsuhiro Taguchi,1,3 Shunsuke Tanigawa,1 Junji Yatsuda,4 Tomomi Kamba,4 Satoru Takahashi,5 Hidetake Kurihara,6 Masashi Mukoyama,2 and Ryuichi Nishinakamura1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Previous research has elucidated the signals required to induce nephron progenitor cells (NPCs) from pluripotent stem cells (PSCs), enabling the generation of kidney organoids. However, selec- tively controlling differentiation of NPCs to podocytes has been a challenge. Methods We investigated the effects of various growth factors in cultured mouse embryonic NPCs during three distinct steps of nephron patterning: from NPC to pretubular aggregate, from the latter to epithelial renal vesicle (RV), and from RV to podocyte. We then applied the findings to human PSC-derived NPCs to establish a method for selective induction of human podocytes. Results Mouse NPC differentiation experiments revealed that phase-specific manipulation of Wnt and Tgf-b 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-b signaling supported domination of the RV proximal domain. Inhibition of Tgf-b signaling in the third phase enriched the podocyte fraction by suppressing development of other nephron lineages. The resul- tant 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 process–like and slit diaphragm–like structures), and showed functional responsiveness to drug-induced injury. Conclusions Elucidation of signals that induce podocytes during the nephron-patterning process enabled 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 offer a new resource for disease modeling and nephrotoxicity testing. J Am Soc Nephrol 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018070747 Podocyte disorders manifest as nephrotic syn- drome and/or glomerulosclerosis, which progress Received July 20, 2018. Accepted December 3, 2018. to renal failure. Thus, growing attention has been Published online ahead of print. Publication date available at paid to podocyte research in recent years.1,2 Owing www.jasn.org. to the poor availability of primary human Correspondence: Dr. Ryuichi Nishinakamura, Department of podocytes, artificially immortalized podocyte cell Kidney Development, Institute of Molecular Embryology and lines3,4 have made great contributions to many Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860- 0811, Japan, or Dr. Atsuhiro Taguchi, Department of Genome podocyte studies. However, these cells do not retain Regulation, Max Planck Institute for Molecular Genetics, Ihnestraße the original characteristics of podocytes, including 63-73, 14195 Berlin, Germany. E-mail: [email protected] abundant expression of slit diaphragm–associated or [email protected] genes and proteins.5 The lack of resources for Copyright © 2019 by the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc, 2019 ISSN : 1046-6673/3002-ccc 1 BASIC RESEARCH www.jasn.org podocytes with sufficient functional characteristics has been a Significance Statement bottleneck in this field. We and others previously developed methods for induction of Recent progress in stem cell biology has enabled researchers to nephron progenitor cells (NPCs) from pluripotent stem cells induce nephron progenitor cells (NPCs) and kidney organoids from 6–9 pluripotent stem cells (PSCs). However, shepherding NPC differ- (PSCs), enabling derivation of kidney organoids. Molecular pro- fi fi entiation toward a speci c nephron segment remains a challenge. ling of the sorted podocytes, comprising approximately 7.5% of The authors observed the effects of various growth factors in cul- the human kidney organoids, confirmed characteristic features that tured mouse embryonic NPCs during three phases of the differ- were shared with murine and human podocytes.10 Recent progress entiation process, demonstrating that phase-specific manipulation in the kidney organoid field has achieved higher-order organiza- of Wnt and Tgf-b signaling is critical for podocyte differentiation. tion.9 However, it remains a challenge to selectively induce Using this insight into the nephron-patterning process, they were able to selectively induce human PSC-derived podocytes with podocytes by controlling the nephron-patterning process from molecular, morphologic, and functional characteristics of human NPCs. Although several groups have reported methods for induc- podocytes. This novel protocol will facilitate accessibility to human tion of podocyte-like cells from human induced PSCs (hiPSCs),11–13 podocytes, and these PSC-derived podocytes are expected to the resultant cells expressed only a few selected marker genes at serve as a valuable resource in kidney research. quite low levels and lacked typical slit diaphragm formation. We fi reasoned that this issue could be addressed by suf cient understand- kindly provided by Dr. Andrew P. McMahon (University of fi ing of the podocyte speci cation process and signaling from NPCs. Southern California). All animal experiments were performed The kidney develops by interactions of ureteric bud (UB) and in accordance with institutional guidelines and approved by metanephric mesenchyme (MM). The MM includes NPCs and the Licensing Committee of Kumamoto University (A29–040). stromal progenitors,14 the former of which express transcription factor Six2 and give rise to epithelial nephrons.15 Wnt signaling Podocyte Induction from Mouse NPCs from the UB triggers condensation of a subset of NPCs below the Metanephroi were isolated from embryonic day (E) 15.5 MafB- UB tip to form the pretubular aggregate (PA), followed by GFP embryos and manually minced in PBS(2)usingforceps. the epithelial renal vesicle (RV).16,17 These steps are designated Minced tissues were dissociated by incubation in 0.25% trypsin- mesenchymal-to-epithelial transition (MET). The RV EDTA at 37°C for 8 minutes. After blocking with normal shows proximodistal polarization, at least by gene expression mouse serum (Thermo Fisher Scientific), dissociated cells levels.18–20 Each part of the RV further elongates along the were stained with anti-Robo2 and anti-Pdgfrb primary anti- proximodistal axis and differentiates into committed nephron bodies for 30 minutes on ice. After secondary antibody staining, 2 2 segments, including podocytes, parietal epithelial cells (PECs), NPCs were sorted as a Robo2high/Pdgfrb /MafB-GFP population proximal tubules (PTs), and distal tubules (DTs). using a FACS SORP Aria (BD Biosciences). NPCs were seeded at Previous genetic studies revealed the requirement and suf- 100,000 cells/150 ml serum-free differentiation medium with vari- ficiency of Wnt signaling for the MET process.16,17,21–23 Ac- ous concentrations of CHIR99021 (CHIR) (Axon Medchem) and cordingly, in vitro experiments demonstrated the sufficiency 10 mM Y27632 (Wako) in 96-well low-cell-binding U-bottom of transient Wnt signaling for MET induction in the isolated plates (Thermo Fisher Scientific). The plates were centrifuged MM.24,25 Furthermore, a recent study showed both promo- (210 3 g, 4 minutes) and cultured at 37°C. After initial CHIR tional and suppressive roles of Wnt signaling during the later induction, aggregated cells were transferred to a 3.0-mmpore phase (after RV formation) of distal and proximal nephron transwell insert (Corning) in serum-free differentiation medium development, respectively.26 However, the patterning mecha- containing Fgf9 (10 mg/ml; R&D Systems) and cultured with the nism for the proximodistal domain of the RV as well as the following factors: CHIR (0.5, 2 mM); IWR-1 (2 mM; Sigma- signals that specify the podocyte lineage during the later pro- Aldrich); activin A (10 ng/ml; R&D Systems); SB431542 (SB) (5, cess of nephron patterning remain to be elucidated. 25, 100 mM; Wako); retinoic acid (RA) (10 mM; Sigma-Aldrich); In this study, we investigated the podocyte lineage–specification BMS493 (10 mM; Tocris Bioscience); Jagged1-Fc (10 mg/ml; R&D factors by dissecting the nephron development process into Systems); ɤ-secretase inhibitor (2 mM; Merck). The culture me- three distinct steps: NPCs to PA, PA to RV,and RV to podocytes. dium was replaced at specified points or every 2 days. Induced For this purpose, we initially employed mouse embryonic NPCs, tissues were harvested for analysis at day 6. The serum-free differ- and then applied the findings in the mouse experiments to entiation medium comprised DMEM/F12 supplemented with 1% hiPSC-derived NPCs to establish a method for selective induction insulin-transferrin-selenium and 1% penicillin-streptomycin of human podocytes. (Thermo Fisher Scientific). Detailed antibody information is pro- vided in Supplemental Table 1. METHODS Cell Culture The NPHS1-GFP knock-in hiPSC line was generated Animals and maintained as described.10 The 201B7 hiPSC line28 was MafB-GFP knock-in (MafB-GFP) mice were described previ- maintained in the same way. The
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