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Manipulation of -Patterning Signals Enables Selective Induction of 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 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 . 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: 304–321, 2019. doi: https://doi.org/10.1681/ASN.2018070747

Podocyte disorders manifest as and/or glomerulosclerosis, which progress to renal Received July 20, 2018. Accepted December 3, 2018. failure. Thus, growing attention has been paid Published online ahead of print. Publication date available at 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 , 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 .5 The lack of resources for Copyright © 2019 by the American Society of

304 ISSN : 1046-6673/3002-304 J Am Soc Nephrol 30: 304–321, 2019 www.jasn.org BASIC RESEARCH 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 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 (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 .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 (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 former line was used to in- ously.27 Six2-GFP-Cre transgenic (Six2-GFP) mice15 were vestigate the induction efficiency, whereas the latter was used

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A B Six2-GFP− cells + 1.00E+00 Six2-GFP NPCs Ab-sorted NPCs

Sorting 1.00E-01

1.00E-02 Pdgfrb

Irrelevant channel 1.00E-03

1.00E-04 Relative mRNA expression Six2 Pax2 Sall1Cited1 Robo2 Robo2 MafB-GFP C 0 h 12–48 h day 6 (analysis) STEP 1 E15.5 MafB-GFP Sorting basal condition NPCs Nephron epithelia mouse kidneys high Robo2 CHIR treatment CHIR 0.5 µM (Podocytes: MafB-GFP+) Pdgfrb− MafB-GFP− D E CHIR treatment 0–24 h 0–48 h ** ** Bright-field MafB-GFP Bright-field MafB-GFP 15 *

10 3 µ M

5 day 6

5 µ M 0 MafB-GFP+ ratio at day 6 (%) 3 µM 5 µM 3 µM 5 µM 24 h 24 h 48 h 48 h F G 24 h 48 h 0 h 1.00E+00 24 h 48 h 1.00E-01

1.00E-02 / DAPI 1.00E-03 Lhx1 / Lhx1

Relative mRNA expression 1.00E-04 Six2 Wnt4 Lhx1 Pax8 Cdh1

Cdh1 NPC PA PA & RV RV H

0 h 24 h 48 h

STEP1 basal NPCs PA RV CHIR 3 µM CHIR 0.5 µM

Figure 1. Optimal duration and strength of Wnt signaling are essential for MET and podocyte differentiation. (A) Flow cytometry analysis of E15.5 MafB-GFP embryonic kidneys with anti-Robo2 and anti-Pdgfrb antibodies (n=3). NPCs were purified as a Robo2high/ 2 2 Pdgfrb /MafB-GFP population. (B) Relative expression levels of NPC-associated genes to b-actin expression (n=3). Blue and gray 2 2 bars, Six2-GFP and Six2-GFP+ cells sorted from E15.5 Six2-GFP embryonic kidneys, respectively; orange bars, Robo2high/Pdgfrb / 2 MafB-GFP NPCs sorted from E15.5 MafB-GFP embryonic kidneys. Ab-sorted, sorted upon antibody-based staining. (C) Schematic

306 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 304–321, 2019 www.jasn.org BASIC RESEARCH for immunohistochemical, TUNEL assay, qRT–PCR, RNA- Collection of Human Adult Podocytes seq, electron microscopy, and western blot analyses, as Human adult kidney tissues were obtained from the normal well as drug-induced injury tests. The RN7 hiPSC line was parts of excised kidney specimens from three patients with established from peripheral of a healthy volunteer using renal cancer under approval from the Ethics Review Commit- Sendai virus vectors as described.29 The detailed characteriza- tee of the Faculty of Life Sciences, Kumamoto University (No. tion of this line will be described elsewhere. The experiments 1050) after receiving informed consent from each patient. The were performed in accordance with the institutional guide- patients were aged 49, 69, and 80 years, and all three had normal lines and approved by the licensing committee of Kumamoto renal function and normal urinalysis findings. The normal University (Nos. 359 and 1453). A conditionally immortalized parts of the kidney specimens were immediately immersed human podocyte cell line was purchased from Bristol Univer- in high- DMEM (Sigma-Aldrich) supplemented sity and cultured as described.4 Briefly, cells were cultured in with 10% FBS on ice. Glomeruli were isolated as de- RPMI 1640 supplemented with 10% FBS at 33°C and passaged scribed32 and sequentially digested with an enzyme mixture every 4–5 days. For induction of the podocyte phenotype, cells of collagenase XI (1 mg/ml) and dispase (2.4 U/ml) at 37°C were cultured at 37°C for 14 days. Cells with fewer than four for 20 minutes, followed by 0.25% trypsin-EDTA at 37°C passages since arrival were used for analyses. for 8 minutes. After the glomerular dissociation, erythro- cytes were hemolyzed with RBC lysis buffer (Thermo Fisher Podocyte Induction from hiPSC-Derived NPCs Scientific). After blocking, the dissociated cells were stained NPCs were induced from hiPSCs as described.9 NPCs were with anti-human and anti-human PODOCALYXIN 2 sorted as an ITGA8+/PDGFRa population using the FACS primary antibodies for 30 minutes on ice. After secondary anti- SORP Aria.30 Sorted human NPCs were seeded at 50,000 or body staining, human adult podocytes were sorted as a 100,000 cells/150 ml serum-free differentiation medium NEPHRIN+/PODOCALYXIN+ population using the FACS containing CHIR (3 mM) and Y27632 (10 mM) in 96-well SORP Aria. low-cell-binding U-bottom plates. The plates were centri- fuged (210 3 g, 4 minutes) and cultured at 37°C. After Drug Injury of Induced Podocytes 24 hours, aggregated cells were transferred to a 3.0-mmpore Induced podocytes in 96-well low-cell-binding U-bottom transwell insert in serum-free differentiation medium containing plates were treated with 30 mg/ml puromycin aminonucleo- Fgf9 (10 mg/ml) and cultured with step 2 factors including IWR-1 side(PAN)(Wako)in200ml DMEM/F12 supplemented with (2 mM), SB (5 mM), and RA (10 mM). At 48 hours after culture 5% FBS, 1% insulin-transferrin-selenium, and 1% penicillin- initiation, differentiating cells on the transwell were transferred to streptomycin. After 48 hours of treatment at 37°C, podocytes fresh medium containing step 3 factors including IWR-1 (2 mM) were harvested and analyzed. and SB (5 mM). The culture medium was replaced at specified points or every 3 days. The induced podocytes were harvested for Statistical Analyses analysis on day 9 or 12. Data are presented as mean6SEM. t test was applied for sta- tistical analyses of differences between two groups. Differ- Kidney Organoid Induction from hiPSC-Derived NPCs ences with values of P,0.05 were considered statistically Kidney organoids were differentiated by coculture of induced significant. NPCs with mouse embryonic spinal cord taken from E12.5 embryos, and culturedat theair–fluid interface onpolycarbonate Data Availability filters (0.8 mm; Whatman) supplied with DMEM/F12 The RNA-seq data have been deposited in the NCBI Bio Sample supplemented with 10% FBS and 1% penicillin-streptomycin.31 database under GEO accession number GSE116471. The culture medium was refreshed every 3 days. The induced A detailed description of methods is included as Supple- organoids were harvested for analyses on day 9. mental Information.

representation of the experiments investigating the initial CHIR treatment of NPCs for MET induction. (D) Bright-field (left panels) and fluorescence (MafB-GFP, right panels) images of induced tissues at day 6 by modulating the concentration and duration of initial CHIR treatment (n=3). Scale bars, 200 mm. (E) Results of flow cytometry analyses at day 6 by modulating the concentration and duration of initial CHIR treatment (n=3). *P,0.05, **P,0.01, t test. (F) Immunostaining for Lhx1 (PA and RV marker) and Cdh1 (RV marker), and nuclear DAPI staining in differentiating cells at the indicated time points (n=3). Low-magnification (upper panels) and high-magnification (lower panels) images are presented. Epithelial vesicles are outlined by yellow dashed lines. Scale bars, 20 mm. (G) Temporal expression levels of stage- specific marker genes at the indicated time points (n=3). Relative expression levels of transcripts to b-actin expression are presented. (H) Schematic representation of the time course for the early stages of differentiation. basal, basal condition. Data are shown as means6SEM.

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A 0 h 24 h 48 h day 6 (analysis) E15.5 MafB-GFP SortingSTEP 1 STEP 2 basal condition NPCs PA RV Nephron epithelia mouse kidneys high µ Robo2 CHIR 3 M CHIR 0.5 µM (Podocytes: MafB-GFP+) Pdgfrb− MafB-GFP− B C 24–48 h 30 ** CHIR 0.5 µM (basal) CHIR 2 µM SB 25

20 ** cell ratio at

+ 15 Bright-field

day 6 (%) 10

day 6 day 5 MafB-GFP 0 SB MafB-GFP

(basal) CHIR 2 µM CHIR 0.5 µM D E

3 20000 * CHIR 0.5 µM (basal) SB 15000 2 **

cell number 10000 + at 48 h at day 6 1 5000 MafB-GFP Relative mRNA expression 0 0 Wt1MafB FoxC2 Pou3f3 Dkk1 Sox9 SB Proximal RV Distal RV (basal) CHIR 2 µM CHIR 0.5 µM F G H 24–48 h Wt1− cells 70 60000 + CHIR 0.5 µM ** Wt1 cells (basal)CHIR 2 µM SB 60 50000 50 ** 40000 40 Wt1 30000 30 n.s. 20000 ** 20 cell ratio at 48 h (%) 48 h + 10 Cell numbers at 48 h 10000 ** Wt1 * 0 0

SB SB Wt1 / DAPI

(basal) (basal)CHIR 2 µM CHIR 2 µM CHIR 0.5 µM CHIR 0.5 µM

Figure 2. Inhibition of Tgf-b signaling in the PA-to-RV differentiation step expands the RV proximal domain. (A) Schematic repre- sentation of the experiments examining the differentiation step from the PA to the RV. (B) Bright-field (upper panels) and fluorescence

308 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 304–321, 2019 www.jasn.org BASIC RESEARCH

RESULTS Inhibition of Tgf-b Signaling in the PA-to-RV Differentiation Step Supports Domination of the RV Optimal Duration and Strength of Wnt Signaling Are Proximal Domain Essential for MET and Podocyte Differentiation Because the RV proximal domain is considered to contain the Weinitiallydevelopedaculturesystemtoenabledifferentiationof precursors of podocytes,19 we hypothesized that directed dif- epithelialized nephrons from purified mouse embryonic NPCs. ferentiation of NPCs into the proximal RV would enhance the For quantitative evaluation of podocyte induction efficiency, we podocyte induction efficiency. Therefore, we examined the used MafB-GFP mice, which specifically express strong GFP effects of growth factors or inhibitors on the PA-to-RV differ- signals in differentiated podocytes.27,33 On the basis of previous entiation step (24–48 hours, step 2; Figure 2A). Consistent 2 2 reports,34,35 we identified the Robo2high/Pdgfrb /MafB-GFP with our earlier findings (Figure 1, D and E), a higher concen- fraction highly enriched with NPCs in the E15.5 kidney tration of CHIR (2 mM) suppressed the ratio and number of (Figure 1A). The purity of this fraction was confirmed using podocytes compared with the basal condition (0.5 mMCHIR) Six2-GFP mice (NPCs: 95.2%60.37%) (Supplemental Figure 1) (Figure 2, B–D). IWR-1, an inhibitor of Wnt/b-catenin sig- and the isolated cells expressed NPC-specific genes at comparable naling, inhibited epithelialization (Supplemental Figure 4B). levels to those in Six2-GFP+ NPCs (Figure 1B). Thus, we isolated In contrast, 25 mM SB, a Tgf-b/SMAD signaling inhibitor, 2 NPCs from MafB-GFP mice as the Robo2high/Pdgfrb /MafB- significantly increased the ratio and final yield of podocytes 2 GFP fraction and used these cells in the following (Figure 2, B–D, Supplemental Figure 4A). All other reagents experiments. investigated, including activin A, RA, BMS493 (a pan-RA re- We next examined the optimal duration and concentration ceptor antagonist), Jagged-1 Fc, and ɤ-secretase inhibitor of Wnt signaling for the initial differentiation of NPCs (step 1). (DAPT; a Notch signal antagonist), failed to increase the For this, NPCs were transiently treated with 1–10 mM CHIR, a podocyte induction efficiency (Supplemental Figure 4, B–D). chemical Wnt agonist, for 12–48 hours and analyzed at day 6 Examination of the gene expression profile at the RV stage (Figure 1C). After the transient treatment, we continuously (48 hours) revealed that SB treatment upregulated proximal administered 0.5 mM CHIR to support tissue growth and de- domain markers such as Wt1, MafB,andFoxC2 (Figure 2E). In fined this as the basal condition (Supplemental Figure 2). contrast, the expression levels of distal RV markers were Macroscopic examination revealed that only treatment with slightly decreased (Figure 2E). A cellular-level analysis showed 3or5mM CHIR for 24 or 48 hours induced epithelial tissue that both the ratio and number of Wt1+ proximal RV cells formation (Supplemental Figure 3). Interestingly, MafB-GFP+ were increased in the SB-treated tissue, whereas 2 mMCHIR podocytes were robustly induced upon treatment with 3 mM treatment decreased proximal RV cells (Figure 2, F–H). CHIR for 24 hours, whereas few podocytes were induced with These results suggest that inhibition of Tgf-b/SMAD sig- 5 mM CHIR or 48 hours of treatment (Figure 1, D and E). naling in the PA-to-RV differentiation step supports domina- Therefore, we concluded that 3 mM CHIR treatment for the tion of the RV proximal domain, thereby increasing the final initial 24 hours was optimal for MET induction and subse- ratio and number of podocytes in the tissue. quent podocyte differentiation. These results suggest that NPCs require optimal strength and duration of Wnt treatment Inhibition of Tgf-b Signaling after the RV Step Enriches for epithelialization, and that the initial Wnt signaling has a the Podocyte Fraction by Suppressing the strong effect on the eventual podocyte formation. Development of Other Nephron Lineages Gene expression profiling and histologic analysis revealed Next, we investigated the factors that control podocyte lineage that the induced cells started to show Lhx1+, Wnt4+,andPax8+ specification after proximalized RV induction (step 3; Figure aggregates at 24 hours, and subsequently formed an epithelial 3A). Continuous administration of SB from 48 hours to day 6 vesicle at 48 hours (Figure 1, F and G). These results indicated significantly increased MafB-GFP+ podocytes (Figure 3, B and that NPCs underwent MET upon transient CHIR treatment, C, Supplemental Figure 5A). Treatment with 2 mMCHIR and that the differentiating cells at 24 and 48 hours correspon- completely abolished podocyte differentiation, whereas treat- ded to the PA and RV stages, respectively (Figure 1H). ment with IWR-1 did not significantly increase the podocyte

(MafB-GFP, lower panels) images of induced tissues at day 6 by modulating the differentiation factors in the PA-to-RV step (n=4). Scale bars, 200 mm. SB, 25 mM. (C) Results of flow cytometry analyses at day 6 by modulating the differentiation factors in the PA-to-RV step (n=4). **P,0.01, t test. (D) Estimated podocyte numbers in induced tissues at day 6 by modulating the differentiation factors in the PA-to-RV step (n=4). MafB-GFP+ cell numbers were obtained by multiplying the percentages of MafB-GFP+ cells and the total cell numbers in the in- duced tissues. *P,0.05, **P,0.01, t test. (E) Relative expression levels of proximal and distal RV marker genes to b-actin expression at 48 hours (n=3). (F) Immunostaining for Wt1 (proximal RV marker) and nuclear DAPI staining in cytocentrifuged differentiating cells at 48 hours (n=3). Scale bars, 25 mm. (G) Percentages of Wt1+ proximal RV cells at 48 hours (n=3). **P,0.01, t test. (H) Estimated numbers of Wt1+ and 2 Wt1 RV cells in differentiating tissues at 48 hours (n=3). Wt1+ cell numbers were obtained by multiplying the percentages of Wt1+ cells and the total cell numbers in the tissues. *P,0.05, **P,0.01; n.s., not significant; t test. Data are shown as means6SEM.

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A day 6 0 h 24 h 48 h (analysis)

E15.5 MafB-GFP Sorting STEP 1 STEP 2 Proximalized STEP 3 Nephron epithelia NPCs PA + mouse kidneys Robo2high CHIR 3 µM SB RV (Podocytes: MafB-GFP ) Pdgfrb− MafB-GFP−

B C 48 h – day 6 ** CHIR 0.5 µM (basal) SB 50

40

30 cell ratio at + Bright-field 20 day 6 (%) day 6 10

MafB-GFP 0

SB MafB-GFP

(basal) CHIR 0.5 µM

D Podocytes PTs DTs n.s. * 30000 40000 15000 n.s. 30000 20000 10000

cell number 20000 + cell number cell number + at day 6 at day 6 at day 6

10000 + 5000 10000 LTL Cdh1

MafB-GFP 0 0 0

SB SB SB

(basal) (basal) (basal) CHIR 0.5 µM CHIR 0.5 µM CHIR 0.5 µM

E Wt1 Nephrin DAPI merge

F Mouse 0 h STEP 124 h STEP 2 48 h STEP 3 day 6 MET induction RV patterning Lineage specification Proximalized NPCs PA Podocytes Wnt Tgfb-iRV Tgfb-i Wt1 Wt1 Wnt4 MafB Six2 MafB Lhx1 Nphs1 FoxC2 Nphs2

310 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 304–321, 2019 www.jasn.org BASIC RESEARCH proportion (Supplemental Figure 5, B and C). These results SB treatment increased the ratio of podocytes (Figure 4C, suggest that strong Wnt signaling suppresses podocyte dif- Supplemental Figure 6D). Administration of IWR-1 and ferentiation, consistent with a previous report.26 Other tested RA in step 2 also increased the proportion of podocytes in factors, including activin A, RA agonist/antagonist, and hiPSC-derived NPCs, and combination of all three factors Notch agonist/antagonist, did not significantly affect the synergistically increased the final podocyte ratio (Figure podocyte proportion (Supplemental Figure 5, B and C). SB 4C). Analysis at the RV stage (48 hours) showed that the com- treatment at this step slightly increased the final number of bination of IWR-1, SB, and RA strongly induced the expression podocytes and decreased the final number of DTs, although of proximal RV markers (Figure 4D) and significantly increased the difference was NS (Figure 3D, Supplemental Figure 5D). the ratio of proximal RV cells in the tissue (Figure 4E). Although Meanwhile, the number of LTL+ PTs was significantly the difference was NS, the estimated number of WT1+ proximal decreased (Figure 3D, Supplemental Figure 5D). A gene ex- RV cells was slightly increased, reflecting the decreased total cell – pression analysis revealed elevated expression of podocyte- number resulting from the dramatic decrease in WT1 nonprox- related genes and decreased expression of PEC-, PT-, and imal RV cells (Figure 4F). Immunohistochemical staining re- DT-related genes (Supplemental Figure 5E). Importantly, vealed that the pHH3+ cell ratio in distal RV cells (JAG1+20) podocytes developed by this high–induction efficiency was decreased (Figure 4G, Supplemental Figure 6E), suggesting method coexpressed a set of typical podocyte-specific pro- suppression of cell proliferation. After RV formation (step 3), the teins (Figure 3E). These results indicate that inhibition of combination of IWR-1 and SB showed maximal induction effi- Tgf-b/SMAD signaling during the nephron segment specifi- ciency for podocytes (Figure 4H, Supplemental Figure 6F). In cation step suppresses the development of other nephron this step, the optimized condition mainly suppressed the differ- segments, resulting in enhanced podocyte induction effi- entiation of LTL+ PTs, whereas no significant change was seen in ciency (Figure 3F). CDH1+ DTs (Figure 4I). When we compared the optimized condition (podocyte condition) with the control condition (cul- Selective Podocyte Induction from hiPSCs ture in the basal condition in step 2 and step 3) side by side Next,wepursuedahigh-efficiency human podocyte induction (Supplemental Figure 7A), the podocyte condition suppressed method from hiPSCs. For this purpose, we employed the the proliferation of PT precursors (JAG1+,20), but not DT pre- NPHS1-GFP line10 and its parental 201B7 line,28 and mainly cursors (POU3F3+,20) (Supplemental Figure 7, B and C). utilized the NPHS1-GFP line for monitoring of podocyte in- TUNEL assays confirmed that the podocyte condition did not duction efficiency. We induced human NPCs by a previously significantly increase cell apoptosis in all three lineages at day 6 described protocol9 andsortedpureNPCsasanITGA8+/ (Supplemental Figure 7, D and E). Wefurther evaluated the gene 2 PDGFRa fraction (Figure 4A, Supplemental Figure 6A).30 expression kinetics during differentiation into podocytes. First, we titrated the concentration and duration of CHIR treat- Podocyte markers were dramatically increased during step 3 ment in the NPC-to-PA phase (step 1) and cultured the cells in (from day 2 to day 9), whereas PT and DT segment markers the basal condition up to day 9 (steps 2 and 3), on the basis of were not upregulated (Supplemental Figure 7F), suggesting the timing of human nephron differentiation.10 The final that our condition selectively allows podocyte differentiation. podocyte induction efficiency at day 9 was examined by We also noted that PECs were absent in the induced podocytes, the NPHS1-GFP+ cellratio.Wefoundthat3 mMCHIRtreat- but detected in the control condition (Supplemental Figure 7, ment for 24 hours was most efficient for podocyte induction GandH).Thefinalized protocol allowed us to induce (Figure 4B, Supplemental Figure 6B). Histologic analysis showed podocytes with .90% purity (Figure 4, H and J), and 2 that NPCs differentiated into the LHX1+/CDH1 PA after produced a two times higher yield of podocytes compared 24 hours of CHIR induction and formed the LHX1+/CDH1+ with the control condition (Supplemental Figure 7I) RVafter 48 hours (Supplemental Figure 6C). During the PA-to- through RV proximalization and subsequent suppression RV differentiation step (step 2), similar to the findings in mice, of the PT lineage.

Figure 3. Inhibition of Tgf-b signaling after the RV step enriches the podocyte fraction by suppressing the differentiation of other nephron lineages. (A) Schematic representation of the experiments examining the differentiation step after the RV step. (B) Bright-field (upper panels) and fluorescence (MafB-GFP, lower panels) images of induced tissues at day 6 by modulating the differentiation factors after the proximalized RV step (n=4). Scale bars, 200 mm. SB, 25 mM. (C) Results of flow cytometry analyses at day 6 by modulating the differentiation factors in the proximalized RV step (n=4). **P,0.01, t test. (D) Estimated cell numbers of podocytes (MafB-GFP+), PTs (LTL+), and DTs (Cdh1+) in induced tissues at day 6 by modulating the differentiation factors after the proximalized RV step (n=4). The cell numbers in each segment were obtained by multiplying the percentages of segment-specific marker-positive cells (see Supple- mental Figure 5D) and the total cell numbers in the induced tissues. *P,0.05; n.s., not significant; t test. (E) Immunostaining for characteristic podocyte markers (Wt1, Nephrin, and Podocin) and nuclear DAPI staining in induced tissues at day 6 (n=3). Scale bars, 10 mm. (F) Model of step-by-step differentiation of mouse NPCs toward podocytes. Data are shown as means6SEM.

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A day 9 0 h 24 h 48 h (analysis) STEP 1 STEP 2 STEP 3 13 days Sorting Metanephric hiPSCs NPCs PA RV Nephron epithelia mesenchyme ITGA8+ PDGFRa– B C D CHIR 0.5 µM (basal) STEP 1 STEP 2 IWR1+SB+RA ** 100 5 ** 40 ** ** 80 ** 4 30 cell ratio

*

cell ratio 60 3 +

+ ** 20

40 at 48 h 2 at day 9 (%) at day 9 (%) 10 20 1 NPHS1-GFP NPHS1-GFP 0 0 Relative mRNA expression 0 3 µM 5 µM 3 µM 5 µM WT1 FOXC2 POU3F3 SOX9 RA 24 h 24 h 48 h 48 h SB IWR1 proximal RV distal RV CHIR treatment (basal) CHIR 0.5 µM IWR1+SB+RA E F G H WT1–cells CHIR 0.5 µM (basal) STEP 3 + ** ** WT1 cells IWR1+SB+RA 90 30000 6 100 ** 80 ** 70 80 60 20000 4 60 50 ** 40 cell ratio at day 9 (%)

40 30 10000 2 + cell ratio at 48 h (%) cell ratio at 48 h (%)

+ + 20 n.s. 20 Cell numbers at 48 h 10 WT1 pHH3 0 0 0 0 NPHS1-GFP SB + ) IWR1 + ) (basal) (basal) Distal RV (basal) IWR1+SB CHIR 0.5 µM CHIR 0.5 µM Proximal(WT1 RV (JAG1 CHIR 0.5 µM IWR1+SB+RA IWR1+SB+RA I J STEP 3 Induced podocytes at day 9

Podocytes PTs DTs Bright-field NPHS1-GFP 50000 10000 2000

n.s. 8000 40000 ** 1500

30000 6000 1000 n.s. cell number at day 9 20000 4000 + cell number at day 9

cell number at day 9 + 500 10000 + 2000 LTL CDH1 0 0 0 NPHS1-GFP

(basal) IWR1+SB (basal) IWR1+SB (basal) IWR1+SB CHIR 0.5 µM CHIR 0.5 µM CHIR 0.5 µM

Figure 4. Step-by-step optimization enables selective podocyte induction from hiPSC-derived NPCs. (A) Schematic representation of the experiments examining podocyte differentiation from hiPSC-derived NPCs. (B) Results of flow cytometry analyses at day 9 by modulating the concentration and duration of the initial CHIR treatment (step 1) (n=5). **P,0.01, t test. (C) Results of flow cytometry analyses at day 9 by modulating the differentiation factors in the PA-to-RV step (step 2) (n=3). SB, 5 mM. *P,0.05, **P,0.01, t test.

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To further test the versatility of the protocol, we applied the We also assessed the global transcriptional profile of same condition to the parental hiPSC line (201B7) and another our induced podocytes compared with hiPSCs, NPCs, hiPSC line derived from a different donor (RN7). We checked immortalized human podocyte cell line cells, and sorted the podocyte induction selectivity by comparison with the human adult podocytes by RNA-seq analysis. The results conventional kidney organoid differentiation method6 that confirmed that many of the signature genes, including slit allows unbiased differentiation of all nephron segments diaphragm–associated genes and core transcriptional fac- (Figure 5A). The kidney organoids derived from each hiPSC tors, were highly expressed and comparable to the levels in line contained around 10%–30% podocytes, whereas the se- human adult podocytes (Table 1). PLA2R1 and THSD7A, lective podocyte induction method achieved 65%–95% effi- recently identified as endogenous antigens responsible for ciency (Figure 5, B and C, Supplemental Figure 8, A–C). The membranous nephropathy,36,37 were moderately ex- resultant tissues were discoid with thicker central regions sim- pressed. Meanwhile, some glomerular basement mem- ilar to conventional kidney organoids, and histologic analysis brane–associated genes were missing in induced podocytes showed that podocytes were distributed throughout the tis- (Table 1). Clustering analyses with previously reported po- sues (Figure 5, D and E). Thus, by optimizing the combination docyte-specific gene entities38,39 revealed significant simi- of factors for each step of the differentiation procedure, we larity between the induced podocytes and human adult succeeded in developing an efficient podocyte induction podocytes (Figure 6, Supplemental Figure 9C). These re- method for multiple hiPSC lines. sults indicate that our induced podocytes have a global signaturegeneexpressionprofile resembling that in hu- Induced Podocytes Exhibit Characteristic Gene man podocytes. Expression Profiles of Human Podocytes The induced podocytes were able to survive and maintain their Induced Podocytes Display Morphologic and characteristics until day 12, but gradually regressed thereafter Functional Characteristics of Podocytes with continuous culture in step 3 medium (Supplemental Fig- Immunohistochemical analyses showed that the induced ure 8D). Thus, we evaluated the gene expression profile of the podocytes expressed WT1 in their nucleus and NEPHRIN induced podocytes at day 12 compared with hiPSCs, immor- on the basolateral cell membrane (Figure 7A). They also talized human podocyte cell line cells,4 and human showed colocalization of NEPH1 and PODOCIN with adult podocytes sorted as a NEPHRIN+/PODOCALYXIN+ NEPHRIN (Figure 7A) and apicobasal polarity with population from sieved human adult kidney glomeruli (Sup- PODOCALYXIN and NEPHRIN expression at the apical plemental Figure 9, A and B) as a genuine positive control. and basolateral regions, respectively (Supplemental Figure Quantitative RT–PCR analyses demonstrated that induced 9D). Transmission electron microscopy showed the pres- podocytes showed high expression levels of podocyte-specific ence of protrusions at the basolateral domain of the genes comparable to those in human adult podocytes (Figure podocytes (Figure 7B), and NEPHRIN was localized 5F). Of note, even in the well established podocyte cell line on the surface membrane of these protrusions (Figure cells, the expression levels of NPHS1 and NPHS2 were quite 7C). Higher magnification observation revealed slit low compared with those in human adult podocytes, whereas diaphragm–like structures recognized as filamentous the expression of SYNPO was maintained to some extent, bridges between adjacent podocytes and an actin lining consistent with a previous report.5 In addition, we detected structure recognized as an electron-dense material at the modest NPHS1 expression in undifferentiated hiPSCs, at cytoplasmic insertion site (Figure 7D). These features re- about 1/1000 the level in human adult podocytes, as reported sembled those of the slit diaphragm precursors we recently previously.11,13 Thus, human podocytes in vivo, which were identified in iPSC-derived conventional kidney organoids not employed in previous studies,11–13 were indispensable as a in vitro.40 positive control to precisely verify the quality of induced Western blotting analyses revealed that the induced podocytes. podocytes abundantly expressed major slit diaphragm–associated

(D) Relative expression levels of proximal and distal RV marker genes to b-actin expression at 48 hours (n=3). (E) Percentages of WT1+ 2 proximal RV cells at 48 hours (n=3). **P,0.01, t test. (F) Estimated numbers of WT1+ and WT1 RV cells in differentiating tissues at 48 hours (n=3). WT1+ cell numbers were obtained by multiplying the percentages of WT1+ cells and the total cell numbers in the tissues. **P,0.01; n.s., not significant; t test. (G) Percentages of pHH3+ proliferating cells within proximal and distal RV cells at 48 hours (n=3). (H) Results of flow cytometry analyses at day 9 by modulating the differentiation factors after the proximalized RV step (step 3) (n=4). SB, 5 mM. **P,0.01, t test. (I) Estimated cell numbers of podocytes (NPHS1-GFP+), PTs (LTL+), and DTs (CDH1+) in induced tissues at day 9 by modulating the differentiation factors after the proximalized RV step (n=3). The cell numbers in each segment were obtained by multi- plying the percentages of segment-specific marker-positive cells and the total cell numbers in the induced tissues. **P,0.01; n.s., not significant; t test. (J) Bright-field (left panel) and fluorescence (NPHS1-GFP, right panel) images of induced podocytes at day 9 (n=4). Scale bars, 200 mm. Data are shown as means6SEM.

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A Conventional kidney organoid induction

0 h day 9 13 days Metanephric mesenchyme Unbiased differentiation hiPSCs Kidney organoids (containing NPCs) (Co-culture with mouse embryonic spinal cord)

Selective podocyte induction STEP 1 STEP 2 STEP 3 0 h MET 24 hRV 48 h Lineage day 9-12 13 days Sorting induction patterning specification Metanephric Proximalized hiPSCs NPCs PA Podocytes mesenchyme RV ITGA8+ Wnt Tgfb-i Tgfb-i PDGFRa− SIX2 LHX1 Wnt-i WT1 Wnt-i WT1 RA FOXC2 NPHS1 NPHS2 SYNPO

BD201B7 hiPSCs C ** Induced podocytes Kidney organoids Podocyte condition + 100

80

60

/PODOCALYXIN 40 cells (%) +

20 PODOCALYXIN 0 NEPHRIN

NEPHRIN Kidney Podocyte organoids condition

E WT1 LTL CDH1 DAPI merge Podocyte condition Kidney organoids

F 1.00E+00 1.00E+00 1.00E+00 1.00E+00 1.00E-01 1.00E-01 1.00E-01 1.00E-01 1.00E-02 1.00E-02 1.00E-03 1.00E-02 1.00E-02 1.00E-03 1.00E-04 1.00E-03 1.00E-03 1.00E-04 1.00E-05 1.00E-04 1.00E-04 1.00E-06 1.00E-05 1.00E-07 1.00E-05 1.00E-05 1.00E-06 1.00E-08 1.00E-06 1.00E-06 Relative mRNA expression NPHS1 NPHS2 WT1 SYNPO hiPSCs Cell line Induced podocytes Human adult podocytes

Figure 5. The established protocol induces human podocytes much more efficiently than the conventional kidney organoid system. (A) Outline of the protocol for selective podocyte induction from hiPSCs compared with the conventional kidney organoid induction. (B and C) Flow cytometry analyses of NEPHRIN and PODOCALYXIN expression (B) and percentages of NEPHRIN+/PODOCALYXIN+ cells (C) at day 9 in kidney organoids and selectively induced podocytes from hiPSCs (201B7) (n=3). **P,0.01, t test. (D) Horizontal (left

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Table 1. Comparisons of podocyte-associated gene expression levels in the RNA-seq analysis Expression Level Gene Symbol hiPSCs Cell Line Induced Podocytes Human Adult Podocytes High-expression genes PODXL 12.30 12.46 4.01 5.90 13.97 13.97 14.49 16.00 15.94 CLIC5 0.00 1.71 0.00 1.07 12.24 12.05 13.11 14.99 14.32 NPHS2 0.00 0.00 0.00 0.00 11.91 11.79 12.47 13.42 13.78 FAT1 10.67 10.55 11.83 11.44 12.20 12.05 12.06 13.55 13.23 NPHS1 3.05 3.20 0.00 0.07 11.54 11.29 10.32 12.36 12.35 ITGB1 10.43 10.13 13.57 13.22 11.54 11.52 12.49 12.49 12.29 SYNPO 3.35 3.85 6.71 7.88 10.28 10.03 9.34 11.66 11.57 MAFB 3.35 3.48 4.63 4.97 12.05 11.99 9.69 11.23 11.41 SULF1 4.40 4.57 3.11 2.07 10.22 9.96 10.64 11.52 11.32 DACH1 2.97 1.39 0.00 0.00 9.95 9.99 9.04 11.95 11.18 WT1 0.65 0.00 0.00 0.00 11.04 10.88 9.54 11.25 10.88 MAGI2 6.79 6.86 5.50 4.71 9.36 9.36 9.43 11.69 10.88 TCF21 0.00 0.00 5.53 4.77 9.79 9.59 9.55 11.42 10.86 FOXC1 0.00 0.00 6.79 6.67 9.35 9.26 9.63 11.17 10.73 LAMA5 3.89 9.11 9.56 10.20 8.72 8.68 9.57 9.76 9.91 COL4A5 7.54 7.54 6.65 6.39 8.83 8.68 8.23 8.95 9.44 BMP7 5.56 5.66 1.69 1.65 8.25 9.14 8.87 9.75 9.28 CD2AP 8.27 8.32 9.68 9.49 8.20 8.16 8.00 10.10 9.13 LMX1B 0.65 0.97 0.00 0.07 7.60 7.26 7.73 8.49 8.50 NPR1 2.23 0.97 0.00 0.00 7.47 7.24 7.08 8.92 8.49 ROBO2 4.87 4.72 1.11 0.07 8.84 9.48 9.30 9.28 7.42 FOXC2 0.00 0.00 2.52 1.70 5.20 7.01 6.58 5.44 7.06 Moderate-expression genes PLA2R1 2.82 3.48 6.65 6.06 9.35 9.37 12.49 14.49 14.11 NPNT 0.65 2.20 4.28 3.65 10.16 10.08 11.92 13.29 13.24 THSD7A 4.51 4.68 3.43 0.00 8.62 9.09 10.38 12.34 12.39 VEGFA 5.74 6.68 7.78 7.70 9.44 9.49 10.96 12.49 12.26 ITGA3 4.91 5.27 11.22 11.56 8.52 8.61 10.80 11.18 11.77 LAMB2 7.11 7.27 8.88 9.39 7.75 7.68 10.94 10.75 11.61 Low-expression genes COL4A3 0.00 0.97 2.69 0.00 0.00 0.00 10.67 12.26 11.58 COL4A4 4.93 4.48 5.19 6.28 1.90 0.00 6.86 11.54 11.09 DDN 0.00 0.00 0.00 2.33 2.90 2.33 8.47 10.35 10.88 Numbers represent normalized read counts. proteins (Figure 7E). They also expressed PLA2R and WT1 To further test the biologic functionality, induced podocytes at much higher levels than immortalized podocyte cell were treated for 48 hours with PAN, used as a model of podocyte line cells. The induced podocytes also showed higher injury and nephrotic syndrome.41–43 Consistent with the expression levels of podocyte-related proteins than phenotype of PAN-induced podocyte injury in vivo,44,45 hiPSC-derived conventional kidney organoids, indicating western blotting analyses showed reduced expression of an advantage of the pure population system for studies on slit diaphragm–associated proteins in PAN-treated low-expression proteins over the heterogeneous organoid podocytes (Figure 7F). Reductions in the phosphorylated system. and mature (upper band) forms of NEPHRIN were also

panel) and coronal (right panel) histologic views of induced podocytes (201B7) at day 9 stained with hematoxylin and eosin (n=3). Scale bars, 200 mm. (E) Immunostaining for podocyte (WT1), proximal (LTL), and distal tubule (CDH1) markers, and nuclear DAPI staining in resultant cells in each condition at day 9 (201B7) (n=3). Scale bars, 100 mm. (F) Results of qRT–PCR analyses for comparisons of podocyte- associated gene expression levels (n=3). Relative expression levels of transcripts to b-actin expression are presented. Cell line, condi- tionally immortalized human podocyte cell line cells; hiPSCs, undifferentiated hiPSCs; human adult podocytes, freshly sorted podocytes 2 from human adult kidney; induced podocytes, selectively induced podocytes from hiPSCs by our protocol; NPCs, ITGA8+/PDGFRa NPCs induced from hiPSCs. Data are shown as means6SEM.

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–2 –1 0 1 2 Row Z–Score

Podxl Rab3b X11.Sep Tmsb4x Nap1l1 Sdc4 Cd59a Pak1 Eif3m Nes Cdkn1c Vegfa Golim4 Adm Cryab Clic3 Rasl11a Tdrd5 Arhgap24 Rhpn1 Shisa3 Hoxc8 Dpp4 Mertk Sema3e Nupr1 Col4a3 Npr3 Igfbp7 Synpo Tcf21 Enpep Npnt Gadd45a lldr2 Ctsl Pard3b Nphs2 Nphs1 Wt1 Tmem150c Pth1r Sema3g Iqgap2 Robo2 Gas1 Mafb Thsd7a Htra1

Cell line hiPSCs NPCs Induced Human adult podocytes podocytes

Figure 6. Induced podocytes have a global signature gene expression profile resembling that in human adult podocytes. Heatmap image and hierarchic clustering of RNA-seq analysis data using the podocyte gene list published by Karaiskos et al.38 Red, upregu- lation; blue, downregulation; cell line, conditionally immortalized human podocyte cell line cells (n=2); hiPSCs, undifferentiated hiPSCs (n=2); human adult podocytes, freshly sorted podocytes from human adult kidney (n=3); induced podocytes, selectively induced 2 podocytes from hiPSCs by our protocol (n=2); NPCs, ITGA8+/PDGFRa NPCs induced from hiPSCs (n=2). observed. Immunohistochemical analyses revealed de- injury46 and patients with nephrotic syndrome.47 Thus, our creased intensity of cell surface–located NEPHRIN (Figure hiPSC-derived podocytes, which show molecular, morphologic, 7G), reminiscent of the decrease/translocation of and functional characteristics of podocytes, will serve as a valu- NEPHRIN observed in an in vivo model of PAN-induced able resource for disease modeling, nephrotoxicity testing.

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WT1/NEPHRIN/ A WT1 NEPHRIN DAPI B

NEPH1/NEPHRIN/ NEPH1 NEPHRIN DAPI C

PODOCIN/NEPHRIN/ PODOCIN NEPHRIN DAPI D

E FG

induced kidney PAN PAN hiPSCs cell lime podocytes organoids －＋ －＋ –250 NEPHRIN NEPHRIN –150 –250 p-NEPHRIN p-NEPHRIN

–150 NEPHRIN NEPH1 PLA2R –150 WT1

NEPH1 PODOCIN –100 WT1 GAPDH WT1 –50

–50 PODOCIN –37

GAPDH –37 NEPHRIN / WT1 DAPI

Figure 7. Induced podocytes can be used for measurement of slit diaphragm–associated proteins upon drug injury. (A) Immunostaining for characteristic podocyte markers (WT1, NEPHRIN, NEPH1, and PODOCIN) and nuclear DAPI staining in induced podocytes at day 12 (n=3). Scale bars, 20 mm. (B) Transmission electron microscopic images of induced podocytes at day 12 (n=2). The black arrowhead in the right panel indicates cellular protrusions extending from the bottom of adjacent induced podocytes. Scale bars, 2 mm (left panel), 500 nm (right panel). (C) Immunoelectron microscopic image of NEPHRIN expression (blue arrows) on the surface of podocyte protrusions (n=2). Scale bars, 200 nm (left panel), 100 nm (right panel). (D) Highly magnified transmission electron micro- scopic images of induced podocytes (n=2). The black arrowheads indicate filamentous bridges between adjacent induced podocytes.

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DISCUSSION lineage in humans, which may be consistent with a previous report that Wnt/b-catenin activity was lowest in the proximal In this study, we have established a highly efficient podocyte end (future podocytes) and weakly present in the future PT induction method from hiPSCs by combining an NPC sorting segment of the S-shaped body.26 We also noted an absence of method and elucidation of the podocyte specification signals. PECs in our induced human podocytes, which is partly con- In the kidney developmental biology field, the signals that sistent with the previous report showing the requirement of dictate the differentiation and patterning of nephron segments Wnt signaling for PEC formation.51 from NPCs have been a major focus of interest. Although in Another signaling pathway that presumably plays a role in vivo genetic studies identified Wnt signaling as the core com- the nephron-patterning process is the Notch pathway,52–56 ponent of the MET process,16,17 these studies had limitations, although it remains controversial whether this signaling especially with regard to time resolution and the roles of sig- has a role, especially for proximal nephron fate specifica- nal intensity and gradient. Meanwhile, in vitro studies have tion.53,56 Indeed, we did not observe significant changes exploited their higher time resolution and more flexible ma- in podocyte induction efficiency after treatment with a nipulation of growth factor signaling.24–26 However, most pre- recombinant Notch agonist in our results. However, vious in vitro studies investigated the bulk embryonic kidney given that a chemical Notch signal agonist has not been well or MM tissue, thereby hampering elucidation of the cell- established, our results may not be strongly conclusive. autonomous signal requirements for the nephron-patterning We observed some differences between murine embryonic process. In this study, we combined the advantages of an in NPCs and hiPSC-derived NPCs. For example, even in the op- vitro directed culture system and purified NPCs to investigate timized condition, murine embryonic NPCs could not differ- the podocyte lineage–specification signals. entiate into podocytes at .50%. Recent studies have shown In the initial step (NPC-to-PA; step 1), optimal strength and that in vivo NPCs are heterogeneous,48,57 whereas our hiPSC- duration of Wnt treatment were critical for MET induction in derived NPCs were considered to be rather homogeneous be- NPCs. Interestingly, among the conditions enabling MET, we cause they do not receive prepatterning signals from UB or found that slight differences in Wnt signal intensity and du- stromal progenitors. Alternatively, it is possible that the NPCs ration during this phase strongly affected the final endowment induced from hiPSCs in our protocol may be somewhat biased of podocytes. This may be partly consistent with a recent study, toward the podocyte lineage compared with those in vivo. which led to a proposed model in which the length of NPC Interestingly, a recent report revealed different developmental exposure to Wnt determines the future proximodistal fate of tendencies of nephron segments among kidney organoid in- nephron epithelia, with NPCs exposed for a shorter time to duction protocols.58 Although species differences could be re- Wnt differentiating into podocytes.48 sponsible for such differences, developmental stage–matched In the second step (PA-to-RV; step 2), we observed that gene profiling of mouse and human embryonic NPCs will be inhibition of Tgf-b/SMAD signaling enriched the RV proxi- indispensable to address these issues. mal domain. A previous report revealed Tgf-b1 expression in We verified the quality of our induced podocytes by exten- the UB tip,49 which is adjacent to the distal side of the RV. This sive gene expression profiling compared with freshly isolated may indicate that Tgf-b/SMAD signaling is a key regulator human podocytes, morphologic analysis, and drug sensitivity. with a role in the patterning process in vivo. It will be inter- Although our podocytes showed proper localization of slit esting to perform genetic loss-of-function studies to prove the diaphragm–associated proteins on the cell membrane, the ex- endogenous role of Tgf-b/SMAD signaling. Inhibition of Wnt pression patterning of NEPHRIN was not sufficiently mature signaling and RA treatment were also effective for enrich- to exhibit a distribution along the , as ment of the RV proximal domain in human NPCs. Because seen in adult podocytes. Electron microscopy showed that RA is known to promote proximal nephron differentiation in the induced podocytes lacked a typical interdigitated struc- zebrafish,50 our results suggest a possible role for RA signaling ture, although slit diaphragm–like filamentous bridges were during the formation of nephron patterning in humans. detected between adjacent podocytes. RNA-seq analyses In the lineage specification step (RV-to-podocytes; step 3), revealed low expression levels of COL4A3 and COL4A4 in inhibition of Tgf-b/SMAD signaling enhanced podocyte in- the induced podocytes compared with human adult duction, mainly by suppressing proliferation of the PT lineage. podocytes. This insufficient maturation as well as the long- Inhibition of Wnt signaling in this step also suppressed the PT term culture limitation of our condition may suggest a

The black arrows in the right panel indicate an actin lining structure in a cellular protrusion of induced podocytes. Scale bars, 200 nm. (E) Western blots of podocyte-associated proteins in hiPSCs, immortalized human podocyte cell line cells, induced podocytes by our protocol, and hiPSC-derived kidney organoids (n=3). The numbers on the right side represent the mol wt of the proteins. (F) Western blots of induced podocytes at 48 hours after PAN treatment compared with nontreated control cells (n=3). (G) Immunostaining for NEPHRIN and WT1, and nuclear DAPI staining in induced podocytes at 48 hours after PAN treatment (n=3). Scale bars, 20 mm.

318 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 304–321, 2019 www.jasn.org BASIC RESEARCH requirement for blood flow and interactions between Supplemental Figure 4. Effects of growth factors or small podocytes and endothelial cells.10 Thus, it will be desirable in the PA-to-RV step on podocyte differentiation. to develop an in vitro podocyte maturation/long-term main- Supplemental Figure 5. Effects of growth factors or small molecules tenance system in the future. When we tried an extended cul- after the RV step on podocyte differentiation. ture of the induced podocytes in a two-dimensional setting, Supplemental Figure 6. Establishment of a selective podocyte in- the cells formed cellular processes with actin bundles and duction method from hiPSC-derived NPCs. maintained WT1 expression for 5 days (Supplemental Figure Supplemental Figure 7. Time-course analyses of differentiating 10, A and B). However, NEPHRIN expression at the mem- cells in the podocyte induction protocol compared with the control brane disappeared by day 2 (Supplemental Figure 10C). These protocol. results indicate that the three-dimensional culture system is Supplemental Figure 8. Highly efficient podocyte induction from a advantageous for maintaining the podocyte characteristics. separate integration-free iPSC line. In summary, our newly developed robust podocyte induc- Supplemental Figure 9. Procedure for isolating human adult po- tion system will facilitate global accessibility to human docytes and RNA-seq analysis of human podocytes. podocytes, and serve as a valuable resource for disease mod- Supplemental Figure 10. Extended two-dimensional culture of the eling and nephrotoxicity testing. induced podocytes. Supplemental Table 1. Antibody information. Supplemental Table 2. Primer sequences.

ACKNOWLEDGMENTS

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AFFILIATIONS

1Department of Kidney Development, Institute of Molecular Embryology and Genetics, and Departments of 2Nephrology and 4Urology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 3Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany; 5Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan; and 6Department of Anatomy and Life Structure, Juntendo University School of Medicine, Tokyo, Japan

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