An efficient intestinal organoid system of direct sorting to evaluate stem cell competition in vitro
Open accessAbstract
Stem cell competition could shed light on the tissue-based quality control mechanism that prevents carcinogenesis. To quantitatively evaluate stem cell competition in vitro, we developed a two-color intestinal organoid forming system. First, we improved a protocol of culturing organoids from intestinal leucine-rich-repeat containing G-protein-coupled receptor 5 (Lgr5)- enhanced green fluorescent protein (EGFP)high stem cells directly sorted on Matrigel without embedding. The organoid-forming potential (OFP) was 25% of Lgr5-EGFP high cells sorted at one cell per well. Using this culture protocol with lineage tracing, we established a two-color organoid culture system by mixing stem cells expressing different fluorescent colors. To analyze stem cell competition, two-color organoids were formed by mixing X-ray-irradiated and non-irradiated intestinal stem cells. In the two-color organoids, irradiated stem cells exhibited a growth disadvantage, although the OFP of irradiated cells alone did not decrease significantly from that of non-irradiated cells. These results suggest that stem cell competition can be evaluated quantitively in vitro using our new system.
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Generating isogenic knockout clones requires single-cell cloning of the organoids, and has been described using FACS sorting.
“Generating isogenic knockout clones requires single-cell cloning of the organoids, and has been described using FACS sorting.4”
From Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9 (2023) · cites this paper as [4]
The Discussion (OFP; page unavailable) describes FACS-based sorting of one stem cell per well and says a single stem cell forms an organoid through clonal proliferation. It does not address isogenic knockout clones or establish that generating them requires single-cell cloning. (LLM, conf=0.87)
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- Authors
- Fujimichi, Y., Otsuka, K., Tomita, M., & Iwasaki, T.
- Affiliations
- Central Research Institute of Electric Power Industry (JP)
- Corresponding author
- Fujimichi, Yuki
- Venue
- Scientific Reports
- Published
- 2019-12-30
- Volume
- 9
- Issue
- 1
- Pages
- 20297-20297
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC6937314
- OpenAlex
- W2998624669
- Cited by
- 38
- References
- 27
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 4 · 2023: 10 · 2022: 7 · 2021: 7 · 2020: 2
- FWCI
- 1.67
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- MEXT | Japan Society for the Promotion of Science (16JK16196)
- Topics
- Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
- Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
- Keywords
- stem cell competition, intestinal organoids, X-ray irradiation, lineage tracing, carcinogenesis prevention, Matrigel culture
- MeSH terms
- Animals, Fluorescent Antibody Technique, Intestines, Organoids, Stem Cells, Biomarkers, Gene Expression, Immunophenotyping, Genes, Reporter, Mice, Knockout, Tissue Engineering, Tissue Culture Techniques, Mice
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