Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 4, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2016806118
Keywords
CRISPR-Cas9; genome-wide screening; Wnt; tissue stem cells; Lgr5
Categories
Funding
- Japan Society for the Promotion of Science [17K07161, 17H06710, 17H03586, 17H01399]
- Agency for Science, Technology and Research (A*STAR)
- National Research Foundation, Prime Minister's Office, 510 Singapore [NRF-NRF12017-03]
- Grants-in-Aid for Scientific Research [17H03586, 17K07161, 17H01399, 17H06710] Funding Source: KAKEN
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Safe utilization of the regenerative potential of adult stem cells for clinical applications requires a comprehensive understanding of the regulatory mechanisms. Epithelial organoid cultures accurately mimic in vivo stem cell-driven epithelial renewal, offering a great ex vivo platform for studying key regulatory mechanisms. Through genome-scale CRISPR knockout screening, Alk, Bclaf3, and Prkra were identified as potential regulators of gastric epithelial differentiation and could suppress stemness/proliferation.
An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3 beta, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.
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