Abstract

Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.

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Acknowledgements

We are grateful to A.A. Arias, J. Puschhof and H. Hu for assistance with mapping of RNA sequencing data; the Franceschi Microscopy and Imaging Center and D.L. Mullendore at Washington State University for TEM preparation and imaging of isolated organoid oocysts; and C. Joo, J. Beumer and O. Kopper for discussions and critical reading of the manuscript. I.H. is the recipient of a VENI grant from the Netherlands Organization for Scientific Research (NWO-ALW, 863.14.002) and was supported by Marie Curie fellowships from the European Commission (Proposal 330571 FP7-PEOPLE-2012-IIF). D.D. is the recipient of a VENI grant from the Netherlands Organization for Scientific Research (NWO-ALW, 016.Veni.171.015). The research leading to these results has received funding from the European Research Council under ERC Advanced Grant Agreement no. 67013 and from NIH NIAIH under R21 AT009174. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society and was funded by a grant from the Dutch Cancer Society.

Author information

Author notes

  1. These authors contributed equally: Inha Heo, Devanjali Dutta.

Affiliations

  1. Hubrecht Institute, Oncode Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), UMC Utrecht, Utrecht, The Netherlands

    • Inha Heo
    • , Devanjali Dutta
    • , Benedetta Artegiani
    • , Norman Sachs
    • , Kim E. Boonekamp
    •  & Hans Clevers
  2. School of Animal and Comparative Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ, USA

    • Deborah A. Schaefer
    •  & Michael W. Riggs
  3. The Maastricht Multimodal Molecular Imaging Institute (M4I), Maastricht University, Maastricht, The Netherlands

    • Nino Iakobachvili
    •  & Peter J. Peters
  4. Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

    • Gregory Bowden
    •  & Roberta O’Connor
  5. Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands

    • Antoni P. A. Hendrickx
    •  & Robert J. L. Willems
  6. Princess Máxima Centre, Utrecht, The Netherlands

    • Hans Clevers

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Contributions

I.H. designed, performed and analysed the experiments and wrote the manuscript. D.D. designed and performed the experiments and helped analyse the data. D.A.S. and M.W.R. performed the mouse experiments. N.I. and P.J.P. performed the TEM experiments. B.A. helped to perform RNA sequencing and analyse the data. N.S. helped in the experiments with lung organoids and immunofluorescence microscopy imaging. K.E.B. helped with the qPCR experiments. R.O. performed oocyst isolation from organoids. G.B. and R.O. performed gene ontology-term analysis of C. parvum genes. A.P.A.H. and R.J.R.W. helped with SEM experiments. R.O. and M.W.R. analysed SEM and TEM data. H.C. and R.O. supervised the project and H.C. wrote the manuscript. All of the authors commented on the manuscript.

Competing interests

N.S. and H.C. are inventors on patents/patent applications related to organoid technology.

Corresponding authors

Correspondence to Roberta O’Connor or Hans Clevers.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7.

  2. Reporting Summary

  3. Supplementary Table 1

    Differentially expressed genes in lung and SI organoids at each time point.

  4. Supplementary Table 2

    Differentially expressed C. parvum genes between 24- and 72-hour post-injection in lung and SI organoids.

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DOI

https://doi.org/10.1038/s41564-018-0177-8