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  • Herein to trace the differentiation and analyze human

    2018-11-14

    Herein, to trace the differentiation and analyze human ES or iPS cell-derived hepatocytes, we carried out gene targeting of a reporter transgene to the ALB locus and established ALB/monomeric Kusabira orange1 (mKO1) knock-in (ALB/mKo1) hES and hiPS cell lines, and report here the use of these knock-in hES and hiPS cell lines, cultured under a modified version of our previously established differentiation procedure (Shiraki et al., 2008b).
    Material and methods
    Results
    Discussion
    Acknowledgments We thank Drs. Nakatsuji and Yamanaka (Kyoto University) for providing hES and hiPS cell lines, Dr. Pedro R. Lowenstein (Cedars-Sinai Medical Center) for providing 293FLPe ampa receptors and FL helper virus, Dr. Neal G. Copeland (National Cancer Institute) for providing the E. coli strains and plasmids for BAC recombineering, and Drs. Takeda and Yusa (Osaka University) for p23-loxP-Zeo plasmid. We thank Ms. Akiko Harada, Yuzuru Iwanaga, and members of the Gene Technology Center at Kumamoto University for technical assistance. This work was supported by a grant (to S.K. and K.M) from the New Energy and Industrial Technology Development Organization (NEDO) and a grant from the National Institute of Biomedical Innovation (to NS). This work was also supported in part by a funding program for Next Generation World-Leading Researchers (NEXT Program) from the Japan Society for the Promotion of Science (JSPS) (to S.K.) and a Global COE grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). S.K. is a global COE member.
    Introduction The maintenance of gastrointestinal homeostasis and the response to tissue damage require the orchestration of intestinal stem and progenitor cell populations. Several signaling pathways regulate the maintenance and differentiation of intestinal stem cells (ISCs) which are also dependent on other cell types, including ISC-derived Paneth cells (PCs) (Barker et al., 2007). PC development within the crypt is dependent on the colony stimulating factor-1 (CSF-1) (Huynh et al., 2009), the primary regulator of the development of macrophages and osteoclasts. CSF-1 effects are mediated by the CSF-1 receptor (CSF-1R), which is expressed on PC, macrophages and osteoclasts (Chitu and Stanley, 2006). In mice deficient in either CSF-1 () or CSF-1R () the PCs fail to develop and the SI exhibited cell proliferation and differentiation defects associated with reduced expression of the ISC marker Lgr5. This phenotype can be rescued in mice by a membrane-spanning cell-surface isoform of CSF-1 that acts locally and does not contribute to the pool of circulating CSF-1, suggesting juxtacrine or paracrine regulation of PC by CSF-1 (Huynh et al., 2009). Consistent with such local regulation, the Csf1 promoter is active in cells in the crypt that closely neighbor PC (Huynh et al., 2009). In single Lgr5+ ISC-derived cultures, there is a close physical association of ISC with PC which express multiple ligands important to ISC maintenance and co-culture of ISC with PC markedly improves organoid formation (Sato et al., 2011). Together, these studies indicated that locally expressed CSF-1 is required for PC development and that PCs directly support the maintenance of ISC. Despite the circumstantial evidence at least two important questions remain unresolved concerning the role of CSF-1 and PC in the regulation of ISC. The first is whether there is a direct or indirect role of macrophages in the lamina propia and/or those below the crypt base that, with PC, are also dramatically reduced in and mice. The second is whether the CSF-1R is required for both PC development and/or PC maintenance in adult mice. To address these questions, we used intestinal epithelial specific inducible deletion of Csf1r and in vitro organoid cultures. We find that the defects evident in mice with germline deletion of Csf1r were recapitulated by the conditional KO studies but the phenotype took several weeks to manifest consistent with the long half life of PC. The use of organoid cultures with either germline or inducible deletion of Csf1r led to reduced colonies and size as well as defective ISC gene expression indicating that CSF-1R is essential and cell intrinsic to the ISC niche.