Title: Organoids: Modeling Development and the Stem Cell Niche in a Dish
Abstract: Organoids are three-dimensional in-vitro-grown cell clusters with near-native microanatomy that arise from self-organizing mammalian pluripotent or adult stem cells. Although monolayer stem cell cultures were established more than 40 years ago, organoid technology has recently emerged as an essential tool for both fundamental and biomedical research. For developmental biologists, organoids provide powerful means for ex vivo modeling of tissue morphogenesis and organogenesis. Here we discuss how organoid cultures of the intestine and other tissues have been established and how they are utilized as an in vitro model system for stem cell research and developmental biology. Organoids are three-dimensional in-vitro-grown cell clusters with near-native microanatomy that arise from self-organizing mammalian pluripotent or adult stem cells. Although monolayer stem cell cultures were established more than 40 years ago, organoid technology has recently emerged as an essential tool for both fundamental and biomedical research. For developmental biologists, organoids provide powerful means for ex vivo modeling of tissue morphogenesis and organogenesis. Here we discuss how organoid cultures of the intestine and other tissues have been established and how they are utilized as an in vitro model system for stem cell research and developmental biology. Mammalian development from a fertilized egg—the first stem cell—to a full-grown body has been a fascinating endeavor for cell and developmental biologists ever since. The emergence of two-dimensional (2D) in vitro cultures of adult epidermal stem cells in the 1970s (Rheinwald and Green, 1975Rheinwald J.G. Green H. 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Successive transformations of an established cell line by polyoma virus and Sv40.Science. 1965; 147: 513-514Crossref PubMed Google Scholar), as a cellular source of—at that time—unknown niche factors. Since their in vitro differentiation potential was rather limited, the cultured putative stem cells had to be transplanted into mice to show that they indeed had the full characteristics of a stem cell: a cell with both capacity to self-renew and multi-lineage differentiation potential (Lajtha, 1979Lajtha L.G. Stem cell concepts.Differentiation. 1979; 14: 23-34Crossref PubMed Google Scholar). Classical transplantation techniques, such as the teratoma assay to assess pluripotent stem cells (Stevens and Little, 1954Stevens L.C. Little C.C. Spontaneous testicular teratomas in an inbred strain of mice.Proc. Natl. Acad. Sci. 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Tech. 2000; 49: 394-406Crossref PubMed Google Scholar). Advances in the understanding of the stem cell niche and regulation of stem cell self-renewal and differentiation led to the first generation of organoid cultures from murine small intestinal stem cells in the late 2000s. Organoid technology allows the establishment of long-term stem cell-based organotypic cultures in the absence of feeder cells by supplementing the culture medium with well-defined stem cell niche factors. A variety of different organoid culture methods have been described over the past years. Organoids can be derived from either pluripotent stem cells (PSCs), i.e., ESCs and induced PSCs (iPSCs), or multipotent organ-specific adult stem cells (ASCs), and be composed of either only epithelial cells or both epithelial and mesenchymal cell types. The rapid advancement of organoid technology promises exciting new insights into developmental biology and opens new avenues for necessary clinical approaches toward treating human disease and regenerative medicine. In this review, we discuss the origin of organoid technology and its use in modeling mammalian development and stem cell differentiation. We highlight the approaches to generate organoids from intestine as well as some other tissues from which such cultures have successfully been derived. We also debate potential applications and existing challenges to be overcome. The term “organoid” was initially used in oncology synonymously with teratomas such as dermoid cysts, and was reported as early as 1946 (Smith and Cochrane, 1946Smith E. Cochrane W.J. CYSTIC ORGANOID TERATOMA: (report of a case).Can. Med. Assoc. J. 1946; 55: 151-152Google Scholar). From the 1960s onward the term was applied to organotypic cultures that self-organized during cell sorting and reaggregation experiments conducted by developmental biologists (Lancaster and Knoblich, 2014Lancaster M.A. Knoblich J.A. Organogenesis in a dish: modeling development and disease using organoid technologies.Science. 2014; 345: 1247125Crossref PubMed Scopus (126) Google Scholar, Weiss and Taylor, 1960Weiss P. Taylor A.C. Reconstitution of complete organs from single-cell suspensions of chick embryos in advanced stages of differentiation.Proc. Natl. Acad. Sci. USA. 1960; 46: 1177-1185Crossref PubMed Google Scholar). With its renaissance not yet ten years ago, the definition of organoids became refined to three-dimensional (3D) in vitro grown structures derived from PSCs or ASCs that self-organize into a near-native microanatomy with organ-specific differentiated cell types and tissue compartmentalization. Requisite for the initial development of the culture system of mouse small intestinal epithelial organoids were several advancements in the understanding of the intestinal stem cell niche. Firstly, the identification of leucine-rich repeat containing G-protein-coupled receptor 5 (Lgr5) as intestinal stem cell-specific marker gene allowing for characterization and purification of these stem cells (Barker et al., 2007Barker N. van Es J.H. Kuipers J. Kujala P. van den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (1878) Google Scholar) and, subsequently, the understanding that adult intestinal stem cells can be both proliferative and long lived in vivo (Barker et al., 2007Barker N. van Es J.H. Kuipers J. Kujala P. van den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (1878) Google Scholar), led to the use of isolated Lgr5+ stem cells as a putative cellular source for organoid cultures. Furthermore, the organoid culture medium containing the niche factors epidermal growth factor (EGF), Noggin, and R-spondin was defined following the discovery of Wnt signaling as an essential cellular signaling pathway for stem cell maintenance in vivo (Korinek et al., 1998Korinek V. Barker N. Moerer P. van Donselaar E. Huls G. Peters P.J. Clevers H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat. Genet. 1998; 19: 379-383Crossref PubMed Scopus (1025) Google Scholar, Pinto et al., 2003Pinto D. Gregorieff A. Begthel H. Clevers H. 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Stem cell proliferation first created cystic spheroids, which then formed crypt-like buddings that within 2 weeks further developed into “mini-guts” with distinct crypt-villus compartmentalization as seen in vivo (Figure 1). The entire epithelium consisted of a monolayer of fully polarized epithelial cells in direct contact with the ECM provided by the Matrigel. The crypt-like structures were composed of Lgr5+ stem cells intermingled in between Paneth cells and, above the crypt bottom, transit-amplifying progenitor cells, while the villus-like domain contained fully differentiated enterocytes. Scattered throughout the organoid epithelium were the other two mature intestinal cell types, goblet cells, and enteroendocrine cells. A recent study by Oudenaarden and co-workers showed by sequencing of mRNA transcripts purified from single cells of intestinal epithelial organoids that a heterogeneous population of cells reflective of the in vivo epithelium is present in these cultures (Grün et al., 2015Grün D. Lyubimova A. Kester L. Wiebrands K. Basak O. Sasaki N. Clevers H. van Oudenaarden A. Single-cell messenger RNA sequencing reveals rare intestinal cell types.Nature. 2015; 525: 251-255Crossref PubMed Scopus (49) Google Scholar). Furthermore, organoids could also be derived from individual intact crypts isolated from the intestinal epithelium by ethylenediaminetetraacetic acid (EDTA) digestion. Against common thought, the study showed that self-organizing near-native intestinal epithelial structures can be built from single stem cells in the absence of a mesenchymal cellular niche (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. 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