MAP(K)ing the Path to Stem Cell Quiescence and the Elusive Enteroendocrine Cell

The existence and interaction of proliferating and quiescent intestinal stem cells have been debated since their discovery in the 1970s. In this issue of Cell Stem Cell, using murine intestinal organoids, Basak et al., 2017Basak O. Beumer J. Wiebrands K. Seno H. van Oudenaarden A. Clevers H. Cell Stem Cell. 2017; 20 (this issue): 177-190Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar induce stem cell quiescence by selective inhibition of EGF/MAPK signaling and define culture conditions that direct differentiation to the enteroendocrine lineage. The existence and interaction of proliferating and quiescent intestinal stem cells have been debated since their discovery in the 1970s. In this issue of Cell Stem Cell, using murine intestinal organoids, Basak et al., 2017Basak O. Beumer J. Wiebrands K. Seno H. van Oudenaarden A. Clevers H. Cell Stem Cell. 2017; 20 (this issue): 177-190Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar induce stem cell quiescence by selective inhibition of EGF/MAPK signaling and define culture conditions that direct differentiation to the enteroendocrine lineage. The existence of different populations of intestinal epithelial stem cells has been debated since the 1970s when actively dividing crypt-based columnar cells (CBCs) (Cheng and Leblond, 1974Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 461-479Crossref PubMed Scopus (539) Google Scholar) and quiescent label-retaining cells (LRCs) (Potten, 1977Potten C.S. Nature. 1977; 269: 518-521Crossref PubMed Scopus (381) Google Scholar) were first identified. Forty years later, the identification of selectively expressed marker proteins permitted transgenic labeling and lineage tracing from these cell populations, demonstrating tissue-appropriate multi-lineage differentiation (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. Clevers H. Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (3952) Google Scholar, Sangiorgi and Capecchi, 2008Sangiorgi E. Capecchi M.R. Nat. Genet. 2008; 40: 915-920Crossref PubMed Scopus (965) Google Scholar). This intensified the debate about the interaction and interdependence of these apparently distinct proliferating and quiescent stem cell populations (Li and Clevers, 2010Li L. Clevers H. Science. 2010; 327: 542-545Crossref PubMed Scopus (934) Google Scholar). In 2013, an important paper from the Winton group used label retention properties to functionally characterize, identify, and segregate the quiescent cell population. This unbiased, stem cell-marker-independent approach identified a population of slowly cycling secretory progenitor cells in the +4 crypt position. These cells continue to express the CBC marker Lgr5 but are committed to secretory lineage differentiation, normally forming mature Paneth and enteroendocrine (EEC) cells. They do, however, retain stem-cell competence and can be recalled to clonogenic capacity in a crypt regenerative setting (Buczacki et al., 2013Buczacki S.J. Zecchini H.I. Nicholson A.M. Russell R. Vermeulen L. Kemp R. Winton D.J. Nature. 2013; 495: 65-69Crossref PubMed Scopus (555) Google Scholar). Now, in this issue of Cell Stem Cell, the Clevers group undertake a comprehensive in vitro assessment of the signaling pathways that regulate proliferative quiescence in Lgr5-expressing cells and then go on to define the culture medium context that directs secretory cell progenitor differentiation to the elusive EEC cell fate (Basak et al., 2017Basak O. Beumer J. Wiebrands K. Seno H. van Oudenaarden A. Clevers H. Cell Stem Cell. 2017; 20 (this issue): 177-190Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The work is undertaken in murine intestinal organoids, an epithelial culture system that retains endogenous differentiation potential but is dependent upon the media supplementation of critical niche-derived factors. Media manipulation allows assessment of how extrinsic intercompartmental and cell-intrinsic morphogen signaling combine in the regulation of intestinal cell fate determination. Basak et al. show that in the presence of sustained Wnt signaling, inhibiting Epidermal Growth Factor (EGF) or downstream Mitogen Associated Protein Kinase (MAPK) pathway activity induced a quiescent state in Lgr5+ stem cells that was readily reversible with resumption of EGF signaling. RNA sequencing showed enrichment of the Buczacki et al. label retaining secretory progenitor signature in quiescent versus proliferative Lgr5-expressing cells, and this included a 7.3-fold rise in expression of chromogranin A—a marker of EEC cells. Given this apparent skew toward EEC cell fate, Basak and colleagues then used combinations of Notch, Wnt, and EGFR/MAPK inhibitors to meticulously define the conditions that favored EEC differentiation. This demonstrated that coordinated inhibition of Wnt, Notch, and EGFR/MAPK massively promoted the differentiation of EEC cells with induction of an EEC cell fate in up to 50% of organoid cells. Comprehensive single-cell transcriptomic analysis showed that organoid culture was able to recapitulate the EEC diversity seen in vivo, with a wide range of identified hormone expression permitting a bioinformatic taxonomic classification of EEC cell type. Interestingly, organoids retained a cell-endogenous “memory” of their intestinal regional identity, as hormone expression varied and was dependent on the position of the tissue source. Thus, organoids derived from the duodenum expressed greater levels of Gastric inhibitory polypeptide (GIP), while distal small intestinal organoids consistently upregulated Neurotensin (NTS) and Glucagon (GCG). This paper is important for several reasons. First, it provides a very valuable addition to the in vivo work of Buczacki et al. demonstrating the inter-relationship of the “active” and “quiescent” stem cell populations in the intestine. Accordingly, a model arises wherein actively dividing Lgr5 CBC stem cells are responsible for daughter cell generation and maintenance of crypt homeostasis. Among the immediate progeny of CBC cell division is a population of quiescent secretory progenitors, usually located at the +4 cell position. In health, these cells are transient and are committed to Paneth or EEC cell fate but act as a functional facultative stem cell pool, capable of reverting back to stem cell function in the event of tissue damage or CBC loss. The work presented by Basak et al. contributes to this model by demonstrating that the conversion of an active Lgr5 cell to a quiescent state is regulated by sustained Wnt signaling, with a concomitant reduction in EGFR/MAPK activity. While the intestinal organoid culture system cannot fully recapitulate the nuanced intercompartmental signaling microenvironment of the intestinal crypt, this is consistent with in vivo findings. The pan Erb-B inhibitor Lrig1 is expressed in quiescent, LRCs and it is conceivable that this mediates a cell-specific reduction in EGFR activity in these cells (Powell et al., 2012Powell A.E. Wang Y. Li Y. Poulin E.J. Means A.L. Washington M.K. Higginbotham J.N. Juchheim A. Prasad N. Levy S.E. et al.Cell. 2012; 149: 146-158Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Second, this paper identifies the contextual requirements necessary to direct Lgr5 cell differentiation toward an EEC cell fate and describes an in vitro system capable of generating large numbers of a diverse population of these cells. Although scarce in vivo, these are a potent and an important intestinal cell type with known or putative roles in the regulation of satiety, intestinal motility, obesity, and depression. The reproducibility of the process defined here allows a readily accessible model for unraveling EEC cell biology, research previously hindered by cell rarity. How such coordinated signaling pathway inhibition is regulated in individual EEC cells in vivo is not clear, but this work illustrates the complexity of context-dependent morphogen regulation and intracellular signal interpretation in the intestine. It is this complexity that allows a limited number of signaling pathways to confer such diverse cellular functions in different cell types and tissues. Finally, this paper touches on the development of neuroendocrine tumors (NETs). Although these are the most common tumors of the small bowel, they are still rare events and research into their pathophysiology has been hindered by the absence of a useful model system. Banck et al., 2013Banck M.S. Kanwar R. Kulkarni A.A. Boora G.K. Metge F. Kipp B.R. Zhang L. Thorland E.C. Minn K.T. Tentu R. et al.J. Clin. Invest. 2013; 123: 2502-2508Crossref PubMed Scopus (208) Google Scholar showed that 29% of small intestinal NETs carry amplifications or activating mutations in the PI3K/AKT/mTOR pathway while other tumors harbor mutations that induce MAPK signaling (BRAF mutation or MEK2 amplification). This implies that the EGF signal necessarily inhibited for physiological EEC cell differentiation is aberrantly reactivated in NET pathogenesis. The organoid culture system described by Basak et al. will be vital in determining how and when this pathological reactivation occurs. This paper builds upon the outstanding work of the Clevers group in this field and significantly enhances our understanding of the dynamic interaction of intestinal stem cell populations and the morphogen signaling pathways that contribute to stem cell quiescence and cell fate determination. Furthermore this paper can be used as a framework to guide the generation of the in vitro tools necessary to answer some of the many questions surrounding the cell biology of the enigmatic EEC cell. Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine CellsBasak et al.Cell Stem CellDecember 8, 2016In BriefBasak et al. identify signals to generate rare enteroendocrine cells (EECs) at high purity through manipulation of intestinal stem cell quiescence. Single-cell sequencing reveals a high level of heterogeneity in hormonal production, which is influenced by the regional identity of the intestinal organoid cultures. Full-Text PDF Open Archive