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Nephron organoids derived from human pluripotent stem cells model kidney development and injury

Nature Biotechnology volume 33, pages 1193–1200 (2015)Cite this article

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Abstract

Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) may enable organ regeneration, disease modeling and drug screening. We report an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+SALL1+WT1+PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+LHX1+ renal vesicles that self-organize into nephron structures. In both two- and three-dimensional culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity.

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Figure 1: Differentiation of hPSCs into posterior intermediate mesoderm.
Figure 2: Differentiation into nephron progenitor cells and spontaneous formation of renal vesicles.
Figure 3: Induction of pretubular aggregates and renal vesicles from nephron progenitor cells.
Figure 4: Self-organizing nephron formation in 2D culture.
Figure 5: Self-organizing nephron formation in 3D culture.
Figure 6: Modeling kidney development and injury in kidney organoids.

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Acknowledgements

The authors thank V. Bijol for providing electron microscopy images of normal human kidneys, L. Racusen (Johns Hopkins Hospital) for HKC-8, J. Barasch (Columbia University) for a mouse ureteric bud cell line, and A.P. McMahon (University of Southern California) for NIH3T3-Wnt4. This study was supported by US National Institutes of Health (NIH) grants R37 DK039773 and R01 DK072381 (J.V.B.); Grant-in-Aid for JSPS (Japan Society for the Promotion of Science); Postdoctoral Fellowship for Research Abroad (R.M.); American Heart Association grant 11FTF7320023 (A.Q.L.); Harvard Stem Cell Institute (A.Q.L., J.V.B. and M.T.V.); and NIH DK102826 and National Kidney Foundation Young Investigator Grant (B.S.F.).

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Authors and Affiliations

  1. Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Ryuji Morizane, Albert Q Lam, Benjamin S Freedman, Seiji Kishi, M Todd Valerius & Joseph V Bonventre

  2. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Ryuji Morizane, Albert Q Lam, Benjamin S Freedman, Seiji Kishi, M Todd Valerius & Joseph V Bonventre

  3. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Albert Q Lam, M Todd Valerius & Joseph V Bonventre

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  1. Ryuji Morizane
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Contributions

R.M. and J.V.B. formulated the strategy for this study. R.M. designed and performed experiments. R.M., A.Q.L. and J.V.B. wrote the manuscript. A.Q.L. and B.S.F. performed nephrotoxicity assays. S.K. performed real-time PCR. M.T.V. and J.V.B. helped to design experiments. All authors helped to interpret the results.

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Correspondence to Ryuji Morizane or Joseph V Bonventre.

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Competing interests

J.V.B. is a co-inventor on KIM-1 patents, which have been licensed by Partners Healthcare to several companies. He has received royalty income from Partners Healthcare. J.V.B. or his family has received income for consulting from companies interested in biomarkers: Sekisui, Millennium, Johnson & Johnson and Novartis.

Integrated supplementary information

Supplementary Figure 1 Metanephric development and published protocols

(a) A schematic illustration of intermediate mesoderm and subsequent differentiation into mesonephros and metanephros. (b) The summary and comparison of published protocols and our new protocol. Takasato et al. Nat Cell Biol. 2014. Taguchi et al. Cell Stem Cell. 2014. RA: retinoic acid.

Supplementary Figure 2 Adjustment of the dose and CHIR treatment time

(a) A schematic illustration of primitive streak and subsequent differentiation into each mesoderm lineage. (b) Pluripotency was evaluated by staining with OCT4 and SOX2 before the differentiation. hESCs differentiated with CHIR 5 μM were positive for T and TBX6 on day 1.5 of differentiation, but cells did not stain for HOXD11. Scale bars: 200 μm. (c) Immunocytochemistry on day 4 of the differentiation with CHIR (3 to 10 μM). Sustained TBX6 expression was observed when the cells were differentiated with high doses of CHIR (7-10 μM). Scale bar: 100 μm. (d) Real-time PCR for MIXL1 in hESCs from day 0 to 7. hESCs were differentiated with CHIR 8 μM for 4 days, and activin 10 ng/ml for 3 days. MIXL1, another marker for primitive streak also showed sustained expression at least until day 4 of the differentiation. Expression returned to very low levels by day 7. n=2. (e) Staining with WT1 and HOXD11 on day 7 of the differentiation. hESCs were differentiated with CHIR 8 μM for 4 days and subsequently with the basic medium (ARPMI) for 3 days. Cells expressed WT1, but not HOXD11. Scale bar 100 μm.

Supplementary Figure 3 Protocol adjustment in hiPSCs

(a) Comparison of CHIR dose in T expression on day 4. A slightly higher dose of CHIR was required for sustained T expression in hiPSCs on day 4. (b) Immunocytochemistry for T, TBX6 or FOXF1 on day 4 of differentiation. hESCs were differentiated with CHIR 8 μM, and hiPSCs were differentiated with CHIR 10 μM. Notably, FOXF1 was negative in hESCs, but was positive in hiPSCs. (c) The tested protocol and representative immunocytochemistry in hiPSCs. Noggin at >5 ng/ml suppressed FOXF1 expression on day 4. To induce WT1 expression on day 7, Noggin 5 ng/ml was optimal. (d) The differentiation protocol and staining for FOXF1 on day 4, and for WT1 on day 7 in hESCs. Either additional Noggin or BMP4 significantly suppressed WT1 expression on day 7, and BMP4 induced FOXF1 expression on day 4, suggesting endogenous BMP4 signal is optimal in hESCs with CHIR treatment alone. (e) The protocol and staining with WT1 and HOXD11 on day 7 in hiPSCs. CHIR 10 μM + Noggin 5 ng/ml followed by activin 10 ng/ml showed the most efficient differentiation into WT1+HOXD11+ cells in hiPSCs. Scale bars: 100 μm.

Supplementary Figure 4 Spontaneous differentiation of SIX2+ cells into nephrons and growth factor screening in 3D culture

(a) Staining with PAX8 and LHX1 in hESCs on day 10 of the differentiation. hESCs were differentiated with CHIR 8 μM for 4 days, activn 10 ng/ml for 3 days, and FGF9 10 ng/ml for 3 days. Sporadic expression of LHX1 was observed in PAX8+ cells. Scale bar: 50 μm. (b) Brighfield imaging for hESCs on day 10 and 21 of the differentiation. FGF9 was withdrawn on day 10, and cells were cultured in the basic medium by day 21. Scale bars: 50 μm. (c) Brightfield and immunocytochemistry for LTL and NPHS1 in hESCs on day 28 of the differentiation. Scale bar: 50 μm. (d) The protocol for growth factor screening. Cells were replated to 3D culture on day 10, and the listed growth factors and small molecules were tested. (e) The number of LTL+ tubules in the organoids. HGF showed a tendency to increase LTL+ tubules. n=2. (f) Brightfield imaging of 3D co-culture.with an ureteric bud cell sphere. Scale bar: 100 μm. (g) Whole-mountstaining for LTL in the organoids on day 16. Scale bar: 100 μm. (h) Immunohistochemistry of the organoids on day 16. Scale bars: 50 μm. LTL: lotus tetragonolobus lectin. NPHS1: Nephrin.

Supplementary Figure 5 Screening for growth factors and small molecules to induce renal vesicles

(a, b) The tested protocols for renal vesicle induction. (c) Immunocytochemistry for SIX2 and LHX1 in structures on day 14 of differentiation. Transient treatment with CHIR 3 μM from day 9 to 11, in combination with FGF9 10 ng/ml from day 7 to 14, increased the number of LHX1+ cells and suppressed SIX2 expression, suggesting mesenchymal epithelial transition. Scale bars: 50 μm. REGM: renal cell growth medium (Lonza, #CC-3190).

Supplementary Figure 6 Kidney development analysis

(a) DAPT was used to suppress Notch signaling from day 14 to 21. (b) CDH1, PODXL and LTL expression on day 21 in cells derived from hESCs in 2D culture. Scale bars: 50 μm. (c) Percentage of LTL+ nephron structures in control and DAPT-treated on day 21. The nephron number was counted as CDH1+ tubules from 10 fields (x20 magnification) of each sample (n=2). CDH1: Cadherin-1 (E-cadherin). PODXL: Podocalyxin-like (Podocalyxin).

Supplementary Figure 7 Nephrotoxic assay

(A) The protocol for the nephrotoxic assay. Gentamicin 5 mg/ml was added from day 21 to 23. (B) Whole-mount staining on day 23 for CDH1, KIM-1 and LTL in kidney organoids derived from hESCs. Scale bars: 50 μm. CDH1: cadherin-1 (E-cadherin). PODXL: podocalyxin-like (Podocalyxin). LTL: lotus tetragonolobus lectin. KIM-1: kidney injury molecule-1.

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Morizane, R., Lam, A., Freedman, B. et al. Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nat Biotechnol 33, 1193–1200 (2015). https://doi.org/10.1038/nbt.3392

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