Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo

  • Cell Stem Cell. 2020 Nov 5;27(5):840-851.e6. doi: 10.1016/j.stem.2020.07.020.
Yi Miao  1 ,  Andrew Ha  2 ,  Wim de Lau  3 ,  Kanako Yuki  4 ,  António J M Santos  4 ,  Changjiang You  5 ,  Maarten H Geurts  3 ,  Jens Puschhof  3 ,  Cayetano Pleguezuelos-Manzano  3 ,  Weng Chuan Peng  6 ,  Ramazan Senlice  7 ,  Carol Piani  7 ,  Jan W Buikema  8 ,  Oghenekevwe M Gbenedio  9 ,  Mario Vallon  4 ,  Jenny Yuan  4 ,  Sanne de Haan  10 ,  Wieger Hemrika  11 ,  Kathrin Rösch  12 ,  Luke T Dang  13 ,  David Baker  13 ,  Melanie Ott  12 ,  Philippe Depeille  8 ,  Sean M Wu  14 ,  Jarno Drost  10 ,  Roeland Nusse  15 ,  Jeroen P Roose  9 ,  Jacob Piehler  5 ,  Sylvia F Boj  7 ,  Claudia Y Janda  16 ,  Hans Clevers  17 ,  Calvin J Kuo  4 ,  K Christopher Garcia  18
Affiliations
  • 1. Department of Molecular and Cellular Physiology, Department of Structural Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
  • 2. Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
  • 3. Oncode Institute, Hubrecht Institute, University Medical Centre Utrecht, Utrecht, the Netherlands.
  • 4. Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA.
  • 5. Division of Biophysics, Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
  • 6. Howard Hughes Medical Institute, Department of Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.
  • 7. Foundation Hubrecht Organoid Technology (HUB), Utrecht, the Netherlands.
  • 8. Department of Cardiology, University Medical Center Utrecht & Utrecht Regenerative Medicine Center, Utrecht University, 3508 GA Utrecht, the Netherlands.
  • 9. Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA.
  • 10. Oncode Institute, Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, the Netherlands.
  • 11. U-Protein Express BV, Yalelaan 62, 3584 CM Utrecht, the Netherlands.
  • 12. Gladstone Institutes and Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
  • 13. Department of Biochemistry, Institute for Protein Design and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98105, USA.
  • 14. Division of Cardiovascular Medicine, Department of Medicine, Cardiovascular Institute and Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
  • 15. Howard Hughes Medical Institute, Department of Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
  • 16. Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.
  • 17. Oncode Institute, Hubrecht Institute, University Medical Centre Utrecht, Utrecht, the Netherlands; Oncode Institute, Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, the Netherlands.
  • 18. Department of Molecular and Cellular Physiology, Department of Structural Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: [email protected].
PMID: 32818433 DOI: 10.1016/j.stem.2020.07.020
Abstract

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in Organoid expansion and single-cell Organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified Organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem Cell Biology.

Keywords
DARPin; Frizzled; Wnt; canonical Wnt signaling; organoids; protein engineering; regenerative medicine; stem cell; surrogate Wnt.
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