Doctoral thesis

Functional characterization and genomic analysis of stem cell organoids derived from adult mouse gallbladder and intestine tissues

ContributorsLugli, Natalia
Defense date2015-12-14

Pioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of organs, including the stomach, small intestine and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R-spondin 1, and is likely facilitated by the fact that, in healthy adults, the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential, and such regeneration is accompanied by the emergence of Lgr5+ stem cells that can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non-regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem cells that can be propagated in culture as organoids for more than a year. The growth of these organoids was stimulated by R-spondin 1 and noggin, and removal of these two growth factors led to partial differentiation towards a hepatocyte lineage. Gallbladder-derived organoids could be transplanted under the liver capsule, where they maintained their architecture for two weeks. Furthermore, single cells prepared from dissociated gallbladder organoids and injected into the mesenteric vein, populated the liver parenchyma of carbon tetrachloride-treated mice. Human gallbladders were also an expandable source of organoid-forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro and be induced to differentiate along the hepatocyte lineage. 3D organoids are not only a great tool for regenerative medicine, but they can also be used to better understand cancer development and progression. For this reason, we sequenced the exomes of organoids derived from single small intestinal crypts of APCmin heterozygous mice. Both normal crypts, heterozygous for APCmin, and transformed crypts, in which the wild-type APC allele was lost, were sequenced. The crypts were isolated from four month old mice. In total, 7 normal crypts 7 and 15 transformed crypts were sequenced; these were derived from three different mice. In the normal crypts we identified 6 single nucleotide substitutions (SNSs), corresponding to 0.86 SNSs per crypt. In the transformed crypts we identified 34 SNSs, corresponding to 2.27 SNSs per crypt. These results indicate that the transformed phenotype is associated with a higher frequency of acquisition of SNSs. Notably, the proliferation rate of the stem cells in the normal and transformed crypts is the same. We conclude that cellular transformation is associated with a higher mutation acquisition rate, consistent with the mutator hypothesis proposed by Larry Loeb. Sequencing of more crypts will help solidify this conclusion.

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Citation (ISO format)
LUGLI, Natalia. Functional characterization and genomic analysis of stem cell organoids derived from adult mouse gallbladder and intestine tissues. 2015. doi: 10.13097/archive-ouverte/unige:80150
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