br Results br Discussion Insertion of reporter cassettes int

Results
Discussion Insertion of reporter cassettes into the Lgr5 locus has enabled characterization of mouse ISCs (Barker et al., 2007, 2010) and development of ex vivo organoid culture systems (Sato et al., 2009). Subsequent transplantation assays using fetal or adult ISCs have paved the way for regenerative therapies of intestinal injuries (Fordham et al., 2013; Yui et al., 2012). However, low cell surface abundance of the LGR5 protein and lack of high-affinity anti-LGR5 next represent a roadblock to isolate human colon stem cells using this marker gene. Our data show that PTK7 is a surrogate of LGR5 in human colon. The methodology developed herein enables the isolation of a cell population largely enriched in CoSCs from human mucosa samples. Remarkably, the possibility of purifying the cell population with highest self-renewal capacity from organoid cultures through PTK7 could facilitate the development of therapeutic approaches based on CoSC transplantation. Identification of a pool of slow cycling ISCs committed to differentiate toward Paneth and enteroendocrine lineages has clarified the long-lasting debate about the existence of quiescent stem cells in the intestine (Clevers, 2013b). One of the defining features of intestinal LRCs is the ability to gain multipotent growth capacity upon tissue injury (Buczacki et al., 2013). However, it remains unclear whether a similar cell type is also present in the human intestine. Expression profiling of PTK7-high cell population suggests the existence of LRC-like cells in the human colonic epithelium and implies phenotypic and functional heterogeneity within the hCoSC compartment similar to that present in mouse crypts. Importantly, Buczacki et al. (2013) demonstrated that mouse LRCs maintain the ability to form intestinal organoids when plated in the culture dish. However, under these conditions, LRCs lose their quiescent/slow-cycling character and revert into actively proliferating ISCs (Buczacki et al., 2013). Similarly, we were unable to identify LRCs in human colon organoids (data not shown). Future efforts should aim to optimize organoid culture conditions in order to promote the concomitant propagation of canonical stem cells and LRCs in vitro. The biology of these reserve stem cells in humans holds great interest as they might be responsible for regeneration of human colon mucosa upon certain insults such as chemotherapy.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction The observation of adult neural stem cells in the mammalian brain (Imayoshi et al., 2008; Reynolds and Weiss, 1992; Zhao et al., 2008) suggested that these stem cells could be mobilized for the repair of the injured or degenerating brain. A growing body of literature shows that adult neural stem cells are recruited in response to neural injury or degeneration, representing an attempt at endogenous repair (Kernie and Parent, 2010; Mitchell et al., 2004). However, this level of endogenous repair was not sufficient to repair the damaged brain. Thus, extensive efforts are underway to harness endogenous neural precursor cells (NPCs) as a novel regenerative therapeutic strategy to treat neural injury or brain degeneration. The recruitment of endogenous adult NPCs involves stimulation of multiple stages of adult NPC development, including proliferation, self-renewal, and differentiation. Thus, an optimal regenerative strategy would stimulate both proliferation/self-renewal and neuronal differentiation in order to generate sufficient numbers of new neurons to replace those lost after brain injury or degeneration. Metformin, an FDA (Food and Drug Administration)-approved diabetes drug, was recently shown to promote adult neurogenesis under both physiological and pathological conditions in vivo (Liu et al., 2014; Jin et al., 2014; Wang et al., 2012). However, metformin has multiple molecular actions (Pernicova and Korbonits, 2014), and it is still not clear which ones are important for its neural effects. For example, metformin activates atypical protein kinase C (aPKC)-mediated CREB-binding protein (CBP) phosphorylation to regulate gluconeogenic gene expression in liver cells and enhance embryonic murine and human NPC differentiation (He et al., 2009; Wang et al., 2012). Moreover, metformin increases the levels of the p53 family member transcription factor TAp73 in cancer cells (Engelmann et al., 2015; Rosenbluth et al., 2008), and TAp73 is essential for adult NPC self-renewal and proliferation (Fujitani et al., 2010), suggesting that this protein might also be important for metformin’s effects in the brain.