Peptide-based Wnt signal activation enables scalable production of clinical-grade patient-derived intestinal organoids for regenerative cell therapy.

“Peptide-based Wnt signal activation enables scalable production of clinical-grade patient-derived intestinal organoids for regenerative cell therapy. Sugihara HY(#)(1), Nagata S(1), Kawasaki S(1), Takahashi J(2), Hiraguri Y(1), Fukuda M(3), Suzuki K(4), Murano T(3), Fujii S(1), Fujii T(1), Shimizu H(1), Ohtsuka K(3), Watanabe M(5), Okamoto R(6), Mizutani T(#)(7). Author information: (1)Department of Gastroenterology and Hepatology, Institute of Science Tokyo, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. (2)Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH, 45229-3026, USA. (3)Endoscopic Unit, Institute of Science Tokyo Hospital, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. (4)Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA. (5)Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo- ku, Tokyo, 113-8421, Japan. (6)Department of Gastroenterology and Hepatology, Institute of Science Tokyo, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. rokamoto.gast@tmd.ac.jp. (7)Department of Gastroenterology and Hepatology, Institute of Science Tokyo, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. tmizutani.gast@tmd.ac.jp. (#)Contributed equally BACKGROUND: Gastrointestinal diseases often involve cellular damage, degeneration or dysfunction in the tract, frequently requiring surgical interventions risking complications and lowered quality of life. Regenerative medicine holds great promise in improving patient care and providing novel treatment options for previously irreparable and untreatable tissues. Despite the clinical potential of intestinal organoids as a resource for regenerative cell therapy and bioengineering, the lack of clinical-grade cultures has hampered further development. Moreover, strategies to efficiently and reliably expand clinical-grade cultures at the scale required for application is limited. METHODS: A GMP-compliant protocol was developed to generate patient-derived colonic organoids from endoscopic biopsies. Clinical-grade colonic organoids cultured and expanded in Type-I collagen were compared to conventional Matrigel cultured organoids. To improve the culture-, cost-, and time-efficiency of culture expansion, several strategies were developed including organoid area-based passaging, one well plate culture, and the incorporation of Wnt activating peptide, PG-008. Conventional recombinant WNT3A culture was compared to the peptide PG-008 culture using single cell RNA sequencing. RESULTS: Clinical-grade collagen cultured organoids exhibited similar culture efficiency to Matrigel. Organoid establishment rate from 60 patients using the GMP-compliant protocol was 82%. The incorporation of PG-008 significantly enhanced organoid growth and stabilized patient-patient variability through intestinal stem cell (ISC) enrichment. Single cell RNA sequencing revealed that PG-008 resulted in remarkably pure culture consisting of ISCs and transit amplifying cells, suitable for rapid and consistent expansion. Intriguingly, our GMP-grade colonic organoids contained LGR5+ ISCs, and injury-induced LGR5- regenerative ISCs, both enriched in peptide culture. CONCLUSIONS: Our study establishes clinical-grade colonic organoids for further application, including autologous transplantations and bioengineering. Further, collagen cultured organoids can be a valuable model facilitating in vitro investigations into regenerative stem cell induction and intestinal regeneration. © 2026. The Author(s). Conflict of interest statement: Declarations. Ethics approval and consent to participate: The study was approved under the project title of “Development of Regenerative Therapy for Intestinal Mucosa Using Cultured Intestinal Epithelial Cells” (M2000-2093, 28th April 2015) by the Ethics Committee of Institute of Science Tokyo. Written informed consent for biopsy collection was obtained from all donors. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.”