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Modelling ciliopathy phenotypes in human tissues derived from pluripotent stem cells with genetically ablated cilia

Nature Biomedical Engineering volume 6, pages 463–475 (2022)Cite this article

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An Author Correction to this article was published on 24 May 2022

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Abstract

The functions of cilia—antenna-like organelles associated with a spectrum of disease states—are poorly understood, particularly in human cells. Here we show that human pluripotent stem cells (hPSCs) edited via CRISPR to knock out the kinesin-2 subunits KIF3A or KIF3B can be used to model ciliopathy phenotypes and to reveal ciliary functions at the tissue scale. KIF3A–/– and KIF3B–/– hPSCs lacked cilia, yet remained robustly self-renewing and pluripotent. Tissues and organoids derived from these hPSCs displayed phenotypes that recapitulated defective neurogenesis and nephrogenesis, polycystic kidney disease (PKD) and other features of the ciliopathy spectrum. We also show that human cilia mediate a critical switch in hedgehog signalling during organoid differentiation, and that they constitutively release extracellular vesicles containing signalling molecules associated with ciliopathy phenotypes. The capacity of KIF3A–/– and KIF3B–/– hPSCs to reveal endogenous mechanisms underlying complex ciliary phenotypes may facilitate the discovery of candidate therapeutics.

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Fig. 1: A CRISPR gene-editing strategy produces stable hPSCs lacking cilia.
Fig. 2: Cilia are critical for efficient terminal differentiation in vivo.
Fig. 3: KIF3A–/– and KIF3B–/– kidney organoids express defects in neuronal and nephron differentiation.
Fig. 4: Modulation of hedgehog pathway activation via kinesin-2 promotes kidney organoid differentiation.
Fig. 5: Smoothened agonist perturbs organoid differentiation similar to ciliary ablation.
Fig. 6: Kinesin-2 knockout organoids express the PKD cystic phenotype.
Fig. 7: Kinesin-2 knockout hPSCs and organoids reveal EV secretion defects in disease-relevant signalling pathways.
Fig. 8: Ciliary function directs morphogenesis in complex tissues derived from hPSCs.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Complete RNA-seq data are available from the NCBI GEO repository under the accession code GSE196807. The raw and analysed datasets generated during the study are too large and complex to be publicly shared (numerous cell lines, replicates, images, blots and experiments, maintained and analysed in specialized file formats and with unique identifiers), yet they are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Cellprofiler and RNA analysis pipelines are available on request from the corresponding author.

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Acknowledgements

We thank S. Shankland, J. Himmelfarb, H. Ruohola-Baker, C. Murry, D. Beier, D. Doherty, J. Scott and M. Bothwell (UW) for helpful discussions; C. Vishy, S. Qi and members of the Freedman and Fu laboratories for assistance with experiments. Studies were supported by an Institute for Stem Cell and Regenerative Medicine Innovation Pilot Award; Lara Nowak-Macklin Research Fund; NIH Awards R01DK117914 (B.S.F.), UG3TR003288 (J.H. and B.S.F.), UG3TR002158 (J.H.), UC2DK126006 (S.S. and B.S.F.), K25HL135432 (H.F.) and U01DK127553 (B.S.F.); the Northwest Kidney Centers; and start-up funds from the University of Washington. Figure 8a was created with Biorender.com.

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Author notes

  1. These authors contributed equally: Nelly M. Cruz, Raghava Reddy.

Authors and Affiliations

  1. Division of Nephrology, University of Washington School of Medicine, Seattle, WA, USA

    Nelly M. Cruz, Raghava Reddy, Christine Tran & Benjamin S. Freedman

  2. Kidney Research Institute, Seattle, WA, USA

    Nelly M. Cruz, Raghava Reddy, Christine Tran & Benjamin S. Freedman

  3. Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA, USA

    Nelly M. Cruz, Raghava Reddy, Christine Tran, Hongxia Fu & Benjamin S. Freedman

  4. Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA

    Nelly M. Cruz, Raghava Reddy, Christine Tran, Hongxia Fu & Benjamin S. Freedman

  5. Department of Biomedical Engineering, Columbia University, New York, NY, USA

    José L. McFaline-Figueroa

  6. Division of Hematology, University of Washington School of Medicine, Seattle, WA, USA

    Hongxia Fu

  7. Department of Bioengineering (Adjunct), University of Washington School of Medicine, Seattle, WA, USA

    Hongxia Fu & Benjamin S. Freedman

  8. Department of Laboratory Medicine and Pathology (Adjunct), University of Washington School of Medicine, Seattle, WA, USA

    Benjamin S. Freedman

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Contributions

N.M.C., R.R., J.L.M.-F., C.T., H.F. and B.S.F. generated and characterized kinesin-2 knockout hPSCs and derived tissues. N.M.C., R.R., H.F. and B.S.F. designed the experiments. B.S.F. wrote the manuscript with revisions from all authors.

Corresponding author

Correspondence to Benjamin S. Freedman.

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Competing interests

B.S.F. is an inventor on patents and patent applications for kidney organoid generation and disease modelling (US10815460B2, United States, 2020-10-27; US2021290632A1, United States, 2021-09-23). The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Maxence Nachury and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Kinesin-2 is dispensable for hPSC morphology and epiblast spheroid formation.

a, Confocal immunofluorescence images of acetylated a-tubulin (AcTub) and DNA in representative fields of undifferentiated control and KIF3A-/- hPSCs. Orthogonal (top) and volume (bottom) views are shown. b, Phase contrast images of representative control and KIF3A-/- epiblast spheroids. c, Quantification (mean ± s.e.m.) of lumen area as percentage of the spheroid area. d-e, Confocal sections showing immunofluorescence for pluripotency, polarity, and ciliary markers in spheroids. A midbody is seen in a KIF3A-/- spheroid (arrow), but not cilia. Scale bars, 25 µm.

Extended Data Fig. 2 Kinesin-2 knockout hPSCs establish a renewable source of diverse human cell types lacking cilia.

a, Neuroepithelial (AcTub), endodermal (α-fetoprotein, AFP), and mesodermal (smooth muscle α-actin, SMA) lineages in EBs. Zoom shows magnification of dashed boxed area with AcTub and DNA intensities increased for clarity. Scale bar, 50 µm. b, Images and c, quantification of pigmented EBs. Scale bar, 500 µm. Knockout (KO) represent pooled data from both KIF3A-/- and KIF3B-/- EBs (mean ± s.e.m., n ≥ 9 independent biological replicates per condition from a total of 8 distinct cell lines). d, Representative images and e, quantification of OCT4 and BRY immunofluorescence intensities in hPSCs after treatment with increasing doses of CHIR99021 in mTeSR1 for 48 hours. Each dot represents a single cell. Data are pooled from three separate experiments. bpp, bits per pixel (raw intensity). Scale bars, 50 µm.

Extended Data Fig. 3 Kinesin-2 knockout tumors exhibit differentiation defects.

a, Representative photographs of whole, unfixed growths retrieved from immunodeficient animals at the same time point. Growths are sliced through their centers to reveal the internal surface. b, A second set of tumors, photographed intact after retrieval from the animals. c-e, Quantification of tissue subtypes within teratomas as a fraction of total area (mean ± s.e.m., n ≥ 5 independent biological replicates per condition from a total of 14 distinct cell lines). f, Ratio of area occupied by SOX2+ cells in TUJ1+ patches of teratoma sections (mean ± s.e.m., n ≥ 4 independent biological replicates per condition from a total of 6 distinct cell lines; *, p = 0.0391).

Extended Data Fig. 4 Hedgehog switching is defective in kinesin-2 knockout organoid cultures.

a, Quantification of SOX2 + area per 96-well of kidney organoid cultures (mean ± s.e.m., n ≥ 3 independent biological replicates per condition from a total of 11 distinct cell lines). b, Wide-field immunofluorescence images of kidney organoid differentiations in a representative 96-well plate. The three left wells contain control cell lines and the three right wells contain KIF3A-/- cell lines. Zoom of boxed regions is shown below each of the wells. Each kidney organoid contains distal tubular (ECAD), proximal tubular (LTL), and podocyte (NPHS1) epithelial cells. Arrow indicates a cluster of ECAD+ cells without proximal tubule and podocyte segments. c, Heatmap of the expression level of genes differentially expressed as a combined function of KIF3A or KIF3B loss in hPSCs (FDR < 0.2). Gene names and labels at right refer to genes associated with enriched gene ontology processes as a function of KIF3B-/- loss in hPSCs in Fig. 4a. d, Representative GLI3 immunoblot of control, KIF3A-/-, and KIF3B-/- undifferentiated hPSCs, with e, band intensity quantification (mean ± s.e.m., n ≥ 3 independent biological replicates per condition from a total of 11 distinct cell lines). f, Band intensity quantification of the blot shown in Fig. 4e, comparing cultures on day 0 to day 18. g, Immunoblot of GLI3 in kidney organoid cultures on days 7, 11, and 18 of differentiation. 3A and 3B indicate KIF3A-/- and KIF3B-/- mutants, respectively. h, Schematic of organoid microdissection (left), with immunoblot of GLI3 on day 18 of differentiation in lysates of whole wells (w), microdissected organoids (o), or remnant stroma (s). i, GLI1 immunoblot of the samples shown in Fig. 4e, comparing cultures on day 0 to day 18.

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Supplementary Video 1

KIF3B–/– cyst grown for several months in suspension culture and transferred into a 250 ml flask.

Supplementary Video 2

Volumetric reconstruction of the lower portion of a cyst derived from KIF3A–/– kidney organoids, showing proximal tubules (LTL, green) and dividing cells (pH3, red), with all nuclei labelled in blue (DAPI).

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Cruz, N.M., Reddy, R., McFaline-Figueroa, J.L. et al. Modelling ciliopathy phenotypes in human tissues derived from pluripotent stem cells with genetically ablated cilia. Nat. Biomed. Eng 6, 463–475 (2022). https://doi.org/10.1038/s41551-022-00880-8

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