Abstract
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-EGFPhigh 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.
Highlights
leucine-rich-repeat containing G-protein-coupled receptor 5 (Lgr5)+ stem cells divide symmetrically, and daughter cells are stochastically destined to retain stemness in niches, or not, by neutral competition to maintain homeostasis of the stem cell pool[4]
We optimized a protocol of culturing organoids from intestinal stem cells highly expressing Lgr5-enhanced green fluorescent protein (Lgr5-EGFPhigh) that were directly sorted on medium containing Matrigel without embedding in Matrigel
We established a high-efficiency organoid culture protocol that could generate an organoid from a single Lgr5-EGFPhigh stem cell by direct sorting into medium containing Matrigel in flat-bottomed plates (Fig. 1A)
Summary
Lgr5+ stem cells divide symmetrically, and daughter cells are stochastically destined to retain stemness in niches, or not, by neutral competition to maintain homeostasis of the stem cell pool[4]. We employed an organoid-forming assay to estimate the survival rate of stem cells exposed to ionizing radiation[11] This assay enabled us to assess the sensitivity of small intestinal stem cells to low-dose radiation. We optimized a protocol of culturing organoids from intestinal stem cells highly expressing Lgr5-enhanced green fluorescent protein (Lgr5-EGFPhigh) that were directly sorted on medium containing Matrigel without embedding in Matrigel. Based on this protocol, we established a high-efficiency system that enabled the quantitative evaluation of stem cell competition between irradiated and non-irradiated Lgr5+ stem cells
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