RRC ID 50020
Author Takahashi Y, Sato S, Kurashima Y, Yamamoto T, Kurokawa S, Yuki Y, Takemura N, Uematsu S, Lai CY, Otsu M, Matsuno H, Osawa H, Mizushima T, Nishimura J, Hayashi M, Yamaguchi T, Kiyono H.
Title A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids.
Journal Stem Cell Reports
Abstract Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.
Volume 10(1)
Pages 314-328
Published 2018-1-9
DOI 10.1016/j.stemcr.2017.11.004
PII S2213-6711(17)30490-3
PMID 29233552
PMC PMC5768885
MeSH Animals Cell Culture Techniques / methods* Epithelial Cells / cytology Epithelial Cells / metabolism* Genetic Vectors Humans Induced Pluripotent Stem Cells / cytology Induced Pluripotent Stem Cells / metabolism* Intestinal Mucosa / cytology Intestinal Mucosa / metabolism* Lentivirus Mice Organoids / cytology Organoids / metabolism* Wnt3A Protein / genetics Wnt3A Protein / metabolism
IF 6.032
Times Cited 21
DNA material CSII-EF-MCS-IRES2-Venus (RDB04384) pCMV-VSV-G-RSV-Rev (RDB04393) pCAG-HIVgp (RDB04394).