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Intestinal stem cells and inflammation
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ScienceDirect Intestinal stem cells and inflammation Samuel Asfaha The intestinal epithelium is renewed every 3–5 days from at least two principal stem cell pools. Actively cycling crypt based columnar (CBC) Lgr5+ cells and slower cycling Bmi1expressing or Krt19-expressing cells maintain the small intestinal and colonic epithelium in homeostasis and injury. Following acute epithelial damage, Lgr5+ stem cells are susceptible to injury and a reserve stem cell or progenitor pool is responsible for regeneration of the epithelium. Current data suggests that intestinal stem cells respond to inflammatory signals to modulate their expansion during epithelial regeneration. Here, we review how inflammation and injury affect intestinal and colonic stem cells. Address University of Western Ontario, Canada Corresponding author: Asfaha, Samuel ([email protected])
Current Opinion in Pharmacology 2015, 25:62–66 This review comes from a themed issue on Gastrointestinal Edited by Nathalie Vergnolle For a complete overview see the Issue Available online 2nd December 2015 http://dx.doi.org/10.1016/j.coph.2015.11.008 1471-4892/# 2015 Elsevier Ltd. All rights reserved.
Introduction The mammalian gastrointestinal tract is lined by a single cell epithelium that serves a variety of important biologic functions including secretion of mucus and defensive proteins, and absorption of nutrients and vitamins. Structurally, the intestinal epithelium is organized into villi and crypts, whereas, the colonic epithelium has crypts, but lacks villi. The gastric epithelium, on the other hand, has a simple columnar epithelium that contains numerous invaginations known as gastric pits that lead into a variety of glandular types (cardiac, fundic and antral).
cells are distinct from the rest of the epithelium in that they are multipotent and capable of self-renewal. Evidence of an intestinal stem cell population was initially provided by tracking inheritance patterns of genetic marks introduced at random into crypt cells [4,5]. Similar results were subsequently obtained in adult human crypts by studying the inheritance patterns of spontaneous mitochondrial mutations [6]. However, these initial studies failed to identify or label specific stem cells within the crypt. Surprisingly, the definitive identification of stem cells within the gastrointestinal tract was only demonstrated as recently as 2007. Through an elegant series of genetic lineage tracing studies carried out by Hans Clevers and colleagues, it was shown that the protein leucine rich repeat containing G-protein coupled receptor-5 (Lgr-5) marks a group of actively cycling stem cells known as the crypt base columnar (CBC) cells [7]. Using Lgr5-GFP-IRES-CreERT2 knock-in mice, Barker et al. (2007) demonstrated that Lgr5 marks long-lived cells that give rise to all the cells within the adult gastric antrum, small intestine and colonic crypts [7,8]. Furthermore, data from clonal lineage tracing of crypt-villus units in combination with mathematical modelling has suggested that Lgr5-positive cells reconstitute all cells of the epithelium via stochastic outcomes of symmetric division [9]. Since the discovery of Lgr5+ cells, several groups have also identified additional stem cell populations in the intestine and colon (Figure 1a) [10–16]. Unfortunately, due to limitations of current lineage tracing technology, it has been difficult to discern whether overlap among these various stem cell markers can explain the observations made to date. Certainly, the findings by Tian et al. (2013) that selective ablation of Lgr5+ stem cells does not alter intestinal homeostasis has strongly supported the notion that more than one stem cell or progenitor pool exists within the intestine [17]. Following Lgr5+ cell ablation, Bmi1+ cells and Krt19+ cells, function as reserve stem cell pools in the intestine and colon, respectively [17,18].
Intestinal stem cells and epithelial injury Remarkably, the gastric antral, intestinal and colonic epithelium are rapidly renewed on a continual basis. Indeed, in the small intestine the cells in the crypt quickly migrate along the crypt-villus axis within 3–5 days [1,2]. The human colonic epithelium is similarly estimated to turnover approximately every 3–4 days [3], and this continual replacement of epithelial cells and regeneration upon injury is believed to originate from stem cells that reside at or near the crypt base. The stem Current Opinion in Pharmacology 2015, 25:62–66
The identification of intestinal stem cell populations using genetic fate mapping techniques has led to considerable excitement about our ability to understand the epithelial response to injury and carcinogenesis. Indeed, the contribution of Lgr5+ stem cells to carcinogenesis was demonstrated by the formation of intestinal adenomas upon mutation of the APC gene in these cells [19]. Lgr5+ stem cells additionally serve as cancer stem cells within established adenomas [20]. www.sciencedirect.com
Stem cells and inflammation Asfaha 63
Figure 1
may contribute to the radio-sensitivity of these cells [25,26]. The role of these factors within the +4 stem cell pool, however, remains unexplored. Thus, the observations to date would suggest that following radiation, epithelial regeneration requires radioresistant reserve stem cell pools to rapidly regenerate actively cycling Lgr5+ stem cells that then allow for proper epithelial regeneration.
(a)
Krt19+ Dll1+ +4 Bmi1, Hopx, mTert Paneth cells Lrig1+
Lgr5+ stem cells
(b)
Promote ISC expansion post injury
Inhibit ISC expansion post injury
Wnt/R-spondin
YAP
Notch
Bmp
JAK/STAT
p53 Current Opinion in Pharmacology
Schematic of intestinal stem cell region and modulating factors or pathways. (a) Schematic diagram of the intestinal crypt demonstrating the location of Lgr5-expressing stem cells in relation to Lrig1+, Krt19+ and +4 stem cells (Bmi1+, Hopx+, mTert+) as well as Dll1+ secretory progenitors. (b) A list of pathways known to modulate intestinal stem cell activity post-epithelial injury.
A number of studies have also examined the effects of epithelial injury or inflammation on ISCs. The majority of these studies have focused on the small intestine rather than the colon, and most of these studies have concentrated on Lgr5+ stem cells rather than cells marked by other stem cell markers. Recognizing these limitations, several studies have attempted to examine the effects of epithelial injury on ISCs, and vice versa, the role of stem cells in the regenerative response. Perhaps the best studied of these injury models is that of radiation injury. Yan et al. (2012) first demonstrated that Lgr5+ stem cells are exquisitively sensitive to high dose radiation injury, whereas, +4 Bmi1+ cells dramatically expand to clonally repopulate the epithelium [21]. More recently, reserve Sox-9+ cells that are also slower-cycling have been shown to respond to epithelial injury following high-dose irradiation [10]. This also appears to be true of the colon where Krt19+ cells located above the crypt base are similarly radioresistant and expand post injury. Nevertheless, the findings of Metcalfe et al. (2014) suggest that Lgr5+ cells are indispensable for intestinal regeneration in the immediate post-radiation period [22]. Thus, Lgr5+ stem cells are radiosensitive, yet remain essential for the rapid regenerative response [23,24]. Both p53, and p53 upregulated modulator of apoptosis (PUMA) are key regulators of apoptotic death of Lgr5+ ISCs after radiation, and www.sciencedirect.com
Interestingly, cellular plasticity also appears important in epithelial regeneration in the setting of inflammation. van Es et al. (2012) showed that Dll1+ secretory progenitors revert back to an active stem cell state following intestinal radiation injury [27]. A number of groups have additionally shown that other relatively quiescent cells may revert to a stem cell state following injury in both the small intestine and colon, allowing for the rapid regeneration of actively cycling Lgr5+ stem cells [12,18,28–30]. During normal homeostasis the gastrointestinal epithelium plays an integral role in the regulation of luminal contents including a diverse microbiota that on occasion can induce epithelial injury and infection. Stem cells within the gastrointestinal tract must therefore, be ready to respond to a variety of antigens, viruses and microbiota. Recent work in this area has revealed Lgr5+ stem cells constitutively express NOD2, an innate immune sensor [31]. NOD2-mediated signaling by microbiota-derived muramyl-dipeptide (MDP) exerts cytoprotection of Lgr5+ stem cells against oxidative stress-mediated cell death [31]. Furthermore, Ly6C+ monocytes suppress stem cell expression and abrogate the response to luminal microbes, suggesting a fascinating relationship between the immune system, microbiota and ISCs [32]. Despite the numerous studies examining stem cell responses to small intestinal injury and more recently the microbiota, there remain few studies examining the effects of inflammation on colonic stem cells. This is particularly important with respect to Inflammatory bowel disease (IBD) where it is believed a multitude of factors ranging from genetics, an altered immune system and the environment (i.e. microbiota) contribute to epithelial injury [33]. Indeed, alterations in the microbiome have been associated with the development of IBD, yet our understanding of how these changes affect ISCs have yet to be defined. The recent ability to culture human intestinal organoids will in the future help us understand the ISC response to inflammation [34]. This culture system provides a novel approach to studying the interaction of bacterial products, host genetic factors and intestinal stem cells [34,35,36,37]. Intriguingly, Davidson et al. (2012) examined the colon of mice post DSS-colitis and reported that ablation of Lgr5+ cells occurs with concurrent loss of crypts [38]. These authors reported mRNA expression of the stem cell markers Ascl2 and Hopx were decreased in the damaged colon, but Lgr5+ stem cells returned back to Current Opinion in Pharmacology 2015, 25:62–66
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normal levels by day 6 after DSS injury. Thus, analogous to the intestine, a reserve stem cell population may be critical for the regeneration of Lgr5+ stem cells during epithelial repair in the colon. Lgr4 may also be important in the colonic stem cell response to injury as Lgr4-deficient mice display dramatically increased susceptibility to DSS-induced colitis in association with reduced stem cell number [39]. From a therapeutic point of view, however, the regeneration of the colonic epithelium from a single Lgr5+ stem cell suggests that functional engraftment and expansion of stem cells may hold promise for treatment of conditions such as IBD [35].
Stem cell regulation in injury A number of signaling pathways are important for the regulation of intestinal stem cells in homeostasis and regeneration. These include the Hedgehog (Hh) and Wingless (Wnt) signaling pathways that are involved in stem cell proliferation during development, regeneration and cancer [23,40–42], and the Bmp and Notch pathways that regulate ISCs during homeostasis and post-injury [41,43–45]. Wnt signaling is the major driving force for homeostatic ISC self-renewal and regeneration in the mammalian intestine, however, restriction of ISC proliferation, particularly during regeneration, is controlled in part, by yes-associated protein 1 (YAP-1) [46]. Barry et al. (2013) reported that YAP halts ISC proliferation by dampening Wnt signaling through restrictions on the nuclear translocation of disheveled [46]. Indeed, YAP is essential for epithelial regeneration following DSS injury, and its regulation important to prevent excessive proliferation that can lead to tumorigenesis [47]. Similarly, Notch promotes Lgr5+ stem cell proliferation in homeostasis and expansion of ISCs post-injury. This is demonstrated by the observation that loss of Notch results in a transient secretory cell hyperplasia associated with reduced Lgr5+ stem cells and impaired crypt regeneration following radiation (Figure 1b) [43]. Other factors such as Snail1 have also been shown to affect ISC fate. Snail1 is expressed by ISCs and plays a role in stem cell maintenance, determining lineage choices and ISC regeneration following radiation damage [44]. Many, if not all, of the above factors and pathways are regulated by cells within the ISC niche including Paneth cells, stromal myofibroblasts, nerves and bone marrow derived inflammatory cells (i.e. macrophages, immature myeloid cells, lymphoid cells, among others) [48]. Paneth cells, for example, are located in close proximity to Lgr5+ stem cells and supply niche signals including Wnt3a, Noggin, Dll4 and EGF [29,49], while mesenchymal cells produce Bmp antagonists [50,51]. Immune cells further secrete inflammatory cytokines such as IL-6 and IL-17 that promote ISC proliferation [48]. During the inflammatory response to injury, a number of additional factors are also involved in the regulation of Current Opinion in Pharmacology 2015, 25:62–66
intestinal stem cells. Important in the response to bacterial products, toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs where its activation in neonates leads to ISC apoptosis through a response linked to ER stress [52]. This ISC apoptotic response has been linked to diseases characterized by ISC loss such as necrotizing enterocolitis. Interestingly, in adults, TLR4 promotes epithelial proliferation and the difference compared to neonates may be related to a developmental change that shifts the TLR4-dependent signaling from pro-apoptotic to pro-proliferative [52,53]. Furthermore, the Jak/Stat pathway has been implicated in the ISC regenerative response. This pathway mediates ISC regeneration in Drosophila, and more recently, Gilbert et al. (2015) showed that in mice, depletion of Stat5 leads to decreased Lgr5+ cell proliferation and impaired crypt regeneration [54–56]. Conversely, Stat5 over-expression increased Lgr5+ cell proliferation and accelerated epithelial regeneration [54–56]. These findings suggest that STAT5 is important in the regulating ISC self-renewal during the inflammatory response.
Summary Thus, during epithelial injury, Lgr5+ stem cells respond to a variety of inflammatory signals to modulate epithelial regeneration. As illustrated in this review, the majority if not all, data on intestinal stem cells responses to injury is focused on Lgr5+ stem cells. Unfortunately, there remains a significant gap in our knowledge of the inflammatory response of other stem cell (i.e. Bmi1+, Lrig1+, Hopx+, among others) populations. Our current understanding of intestinal stem cell responses has been closely linked to our ability to visualize and isolate these stem cells using the described cell markers [11–16]. In this regard, the Lgr5GFP-IRES-CreERT2 mouse first described by Barker et al. (2007) has proven the most robust, and thus made it the easiest to study. This has in large part, been due to the fact that GFP transcription from the Lgr5 locus not only accurately reflects Lgr5 mRNA expression, but also occurs from the same transcript that allows for Cre-mediated lineage tracing [7,57,58,59]. Hence, the cells that express GFP are, for the most part, also the same cells that show stemness, and therefore genetic lineage tracing ability. Unfortunately, this has not always proven to be the case for other established stem cell markers. For instance, FACS isolation of GFP+ cells from Bmi1-expressing cells is only possible from a Bmi1-GFP reporter mouse that was generated using a slightly different genetic strategy than the Bmi1-CreERT2 mouse commonly used for lineage tracing. Moreover, the endogenous Bmi1 gene is ‘‘knocked-out’’ in the Bmi1-GFP reporter mouse, whereas left intact in the Bmi1-CreERT2 mouse [11,60]. Such differences have made it difficult to prove that the cells expressing GFP and CreERT are identical. Furthermore, challenges in our ability to culture and expand non-Lgr5+ stem cell populations have significantly limited our www.sciencedirect.com
Stem cells and inflammation Asfaha 65
understanding of additional stem cell pools in homeostasis and injury. Future studies of progenitor and/or non-Lgr5+ stem cell pools during epithelial injury will allow discovery of factors that regulate additional stem cell responses. Moreover, as our ability to culture and expand all ISC populations improves, we hope that the therapeutic benefit of ISCs will become a reality.
13. Ramalingam S, Daughtridge GW, Johnston MJ, Gracz AD, Magness ST: Distinct levels of sox9 expression mark colon epithelial stem cells that form colonoids in culture. Am J Physiol Gastrointest Liver Physiol 2012, 302:G10-G20.
Conflict of interest
16. Montgomery RK, Carlone DL, Richmond CA, Farilla L, Kranendonk ME, Henderson DE, Baffour-Awuah NY, Ambruzs DM, Fogli LK, Algra S, Breault DT: Mouse telomerase reverse transcriptase (mtert) expression marks slowly cycling intestinal stem cells. Proc Natl Acad Sci USA 2011, 108:179-184.
Nothing declared.
Acknowledgements Dr. Samuel Asfaha is supported by a CIHR Clinician Scientist Phase I Award and CIHR Operating Grant.
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Current Opinion in Pharmacology 2015, 25:62–66
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ScienceDirect Intestinal stem cells and inflammation Samuel Asfaha The intestinal epithelium is renewed every 3–5 days from at least two principal stem cell pools. Actively cycling crypt based columnar (CBC) Lgr5+ cells and slower cycling Bmi1expressing or Krt19-expressing cells maintain the small intestinal and colonic epithelium in homeostasis and injury. Following acute epithelial damage, Lgr5+ stem cells are susceptible to injury and a reserve stem cell or progenitor pool is responsible for regeneration of the epithelium. Current data suggests that intestinal stem cells respond to inflammatory signals to modulate their expansion during epithelial regeneration. Here, we review how inflammation and injury affect intestinal and colonic stem cells. Address University of Western Ontario, Canada Corresponding author: Asfaha, Samuel ([email protected])
Current Opinion in Pharmacology 2015, 25:62–66 This review comes from a themed issue on Gastrointestinal Edited by Nathalie Vergnolle For a complete overview see the Issue Available online 2nd December 2015 http://dx.doi.org/10.1016/j.coph.2015.11.008 1471-4892/# 2015 Elsevier Ltd. All rights reserved.
Introduction The mammalian gastrointestinal tract is lined by a single cell epithelium that serves a variety of important biologic functions including secretion of mucus and defensive proteins, and absorption of nutrients and vitamins. Structurally, the intestinal epithelium is organized into villi and crypts, whereas, the colonic epithelium has crypts, but lacks villi. The gastric epithelium, on the other hand, has a simple columnar epithelium that contains numerous invaginations known as gastric pits that lead into a variety of glandular types (cardiac, fundic and antral).
cells are distinct from the rest of the epithelium in that they are multipotent and capable of self-renewal. Evidence of an intestinal stem cell population was initially provided by tracking inheritance patterns of genetic marks introduced at random into crypt cells [4,5]. Similar results were subsequently obtained in adult human crypts by studying the inheritance patterns of spontaneous mitochondrial mutations [6]. However, these initial studies failed to identify or label specific stem cells within the crypt. Surprisingly, the definitive identification of stem cells within the gastrointestinal tract was only demonstrated as recently as 2007. Through an elegant series of genetic lineage tracing studies carried out by Hans Clevers and colleagues, it was shown that the protein leucine rich repeat containing G-protein coupled receptor-5 (Lgr-5) marks a group of actively cycling stem cells known as the crypt base columnar (CBC) cells [7]. Using Lgr5-GFP-IRES-CreERT2 knock-in mice, Barker et al. (2007) demonstrated that Lgr5 marks long-lived cells that give rise to all the cells within the adult gastric antrum, small intestine and colonic crypts [7,8]. Furthermore, data from clonal lineage tracing of crypt-villus units in combination with mathematical modelling has suggested that Lgr5-positive cells reconstitute all cells of the epithelium via stochastic outcomes of symmetric division [9]. Since the discovery of Lgr5+ cells, several groups have also identified additional stem cell populations in the intestine and colon (Figure 1a) [10–16]. Unfortunately, due to limitations of current lineage tracing technology, it has been difficult to discern whether overlap among these various stem cell markers can explain the observations made to date. Certainly, the findings by Tian et al. (2013) that selective ablation of Lgr5+ stem cells does not alter intestinal homeostasis has strongly supported the notion that more than one stem cell or progenitor pool exists within the intestine [17]. Following Lgr5+ cell ablation, Bmi1+ cells and Krt19+ cells, function as reserve stem cell pools in the intestine and colon, respectively [17,18].
Intestinal stem cells and epithelial injury Remarkably, the gastric antral, intestinal and colonic epithelium are rapidly renewed on a continual basis. Indeed, in the small intestine the cells in the crypt quickly migrate along the crypt-villus axis within 3–5 days [1,2]. The human colonic epithelium is similarly estimated to turnover approximately every 3–4 days [3], and this continual replacement of epithelial cells and regeneration upon injury is believed to originate from stem cells that reside at or near the crypt base. The stem Current Opinion in Pharmacology 2015, 25:62–66
The identification of intestinal stem cell populations using genetic fate mapping techniques has led to considerable excitement about our ability to understand the epithelial response to injury and carcinogenesis. Indeed, the contribution of Lgr5+ stem cells to carcinogenesis was demonstrated by the formation of intestinal adenomas upon mutation of the APC gene in these cells [19]. Lgr5+ stem cells additionally serve as cancer stem cells within established adenomas [20]. www.sciencedirect.com
Stem cells and inflammation Asfaha 63
Figure 1
may contribute to the radio-sensitivity of these cells [25,26]. The role of these factors within the +4 stem cell pool, however, remains unexplored. Thus, the observations to date would suggest that following radiation, epithelial regeneration requires radioresistant reserve stem cell pools to rapidly regenerate actively cycling Lgr5+ stem cells that then allow for proper epithelial regeneration.
(a)
Krt19+ Dll1+ +4 Bmi1, Hopx, mTert Paneth cells Lrig1+
Lgr5+ stem cells
(b)
Promote ISC expansion post injury
Inhibit ISC expansion post injury
Wnt/R-spondin
YAP
Notch
Bmp
JAK/STAT
p53 Current Opinion in Pharmacology
Schematic of intestinal stem cell region and modulating factors or pathways. (a) Schematic diagram of the intestinal crypt demonstrating the location of Lgr5-expressing stem cells in relation to Lrig1+, Krt19+ and +4 stem cells (Bmi1+, Hopx+, mTert+) as well as Dll1+ secretory progenitors. (b) A list of pathways known to modulate intestinal stem cell activity post-epithelial injury.
A number of studies have also examined the effects of epithelial injury or inflammation on ISCs. The majority of these studies have focused on the small intestine rather than the colon, and most of these studies have concentrated on Lgr5+ stem cells rather than cells marked by other stem cell markers. Recognizing these limitations, several studies have attempted to examine the effects of epithelial injury on ISCs, and vice versa, the role of stem cells in the regenerative response. Perhaps the best studied of these injury models is that of radiation injury. Yan et al. (2012) first demonstrated that Lgr5+ stem cells are exquisitively sensitive to high dose radiation injury, whereas, +4 Bmi1+ cells dramatically expand to clonally repopulate the epithelium [21]. More recently, reserve Sox-9+ cells that are also slower-cycling have been shown to respond to epithelial injury following high-dose irradiation [10]. This also appears to be true of the colon where Krt19+ cells located above the crypt base are similarly radioresistant and expand post injury. Nevertheless, the findings of Metcalfe et al. (2014) suggest that Lgr5+ cells are indispensable for intestinal regeneration in the immediate post-radiation period [22]. Thus, Lgr5+ stem cells are radiosensitive, yet remain essential for the rapid regenerative response [23,24]. Both p53, and p53 upregulated modulator of apoptosis (PUMA) are key regulators of apoptotic death of Lgr5+ ISCs after radiation, and www.sciencedirect.com
Interestingly, cellular plasticity also appears important in epithelial regeneration in the setting of inflammation. van Es et al. (2012) showed that Dll1+ secretory progenitors revert back to an active stem cell state following intestinal radiation injury [27]. A number of groups have additionally shown that other relatively quiescent cells may revert to a stem cell state following injury in both the small intestine and colon, allowing for the rapid regeneration of actively cycling Lgr5+ stem cells [12,18,28–30]. During normal homeostasis the gastrointestinal epithelium plays an integral role in the regulation of luminal contents including a diverse microbiota that on occasion can induce epithelial injury and infection. Stem cells within the gastrointestinal tract must therefore, be ready to respond to a variety of antigens, viruses and microbiota. Recent work in this area has revealed Lgr5+ stem cells constitutively express NOD2, an innate immune sensor [31]. NOD2-mediated signaling by microbiota-derived muramyl-dipeptide (MDP) exerts cytoprotection of Lgr5+ stem cells against oxidative stress-mediated cell death [31]. Furthermore, Ly6C+ monocytes suppress stem cell expression and abrogate the response to luminal microbes, suggesting a fascinating relationship between the immune system, microbiota and ISCs [32]. Despite the numerous studies examining stem cell responses to small intestinal injury and more recently the microbiota, there remain few studies examining the effects of inflammation on colonic stem cells. This is particularly important with respect to Inflammatory bowel disease (IBD) where it is believed a multitude of factors ranging from genetics, an altered immune system and the environment (i.e. microbiota) contribute to epithelial injury [33]. Indeed, alterations in the microbiome have been associated with the development of IBD, yet our understanding of how these changes affect ISCs have yet to be defined. The recent ability to culture human intestinal organoids will in the future help us understand the ISC response to inflammation [34]. This culture system provides a novel approach to studying the interaction of bacterial products, host genetic factors and intestinal stem cells [34,35,36,37]. Intriguingly, Davidson et al. (2012) examined the colon of mice post DSS-colitis and reported that ablation of Lgr5+ cells occurs with concurrent loss of crypts [38]. These authors reported mRNA expression of the stem cell markers Ascl2 and Hopx were decreased in the damaged colon, but Lgr5+ stem cells returned back to Current Opinion in Pharmacology 2015, 25:62–66
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normal levels by day 6 after DSS injury. Thus, analogous to the intestine, a reserve stem cell population may be critical for the regeneration of Lgr5+ stem cells during epithelial repair in the colon. Lgr4 may also be important in the colonic stem cell response to injury as Lgr4-deficient mice display dramatically increased susceptibility to DSS-induced colitis in association with reduced stem cell number [39]. From a therapeutic point of view, however, the regeneration of the colonic epithelium from a single Lgr5+ stem cell suggests that functional engraftment and expansion of stem cells may hold promise for treatment of conditions such as IBD [35].
Stem cell regulation in injury A number of signaling pathways are important for the regulation of intestinal stem cells in homeostasis and regeneration. These include the Hedgehog (Hh) and Wingless (Wnt) signaling pathways that are involved in stem cell proliferation during development, regeneration and cancer [23,40–42], and the Bmp and Notch pathways that regulate ISCs during homeostasis and post-injury [41,43–45]. Wnt signaling is the major driving force for homeostatic ISC self-renewal and regeneration in the mammalian intestine, however, restriction of ISC proliferation, particularly during regeneration, is controlled in part, by yes-associated protein 1 (YAP-1) [46]. Barry et al. (2013) reported that YAP halts ISC proliferation by dampening Wnt signaling through restrictions on the nuclear translocation of disheveled [46]. Indeed, YAP is essential for epithelial regeneration following DSS injury, and its regulation important to prevent excessive proliferation that can lead to tumorigenesis [47]. Similarly, Notch promotes Lgr5+ stem cell proliferation in homeostasis and expansion of ISCs post-injury. This is demonstrated by the observation that loss of Notch results in a transient secretory cell hyperplasia associated with reduced Lgr5+ stem cells and impaired crypt regeneration following radiation (Figure 1b) [43]. Other factors such as Snail1 have also been shown to affect ISC fate. Snail1 is expressed by ISCs and plays a role in stem cell maintenance, determining lineage choices and ISC regeneration following radiation damage [44]. Many, if not all, of the above factors and pathways are regulated by cells within the ISC niche including Paneth cells, stromal myofibroblasts, nerves and bone marrow derived inflammatory cells (i.e. macrophages, immature myeloid cells, lymphoid cells, among others) [48]. Paneth cells, for example, are located in close proximity to Lgr5+ stem cells and supply niche signals including Wnt3a, Noggin, Dll4 and EGF [29,49], while mesenchymal cells produce Bmp antagonists [50,51]. Immune cells further secrete inflammatory cytokines such as IL-6 and IL-17 that promote ISC proliferation [48]. During the inflammatory response to injury, a number of additional factors are also involved in the regulation of Current Opinion in Pharmacology 2015, 25:62–66
intestinal stem cells. Important in the response to bacterial products, toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs where its activation in neonates leads to ISC apoptosis through a response linked to ER stress [52]. This ISC apoptotic response has been linked to diseases characterized by ISC loss such as necrotizing enterocolitis. Interestingly, in adults, TLR4 promotes epithelial proliferation and the difference compared to neonates may be related to a developmental change that shifts the TLR4-dependent signaling from pro-apoptotic to pro-proliferative [52,53]. Furthermore, the Jak/Stat pathway has been implicated in the ISC regenerative response. This pathway mediates ISC regeneration in Drosophila, and more recently, Gilbert et al. (2015) showed that in mice, depletion of Stat5 leads to decreased Lgr5+ cell proliferation and impaired crypt regeneration [54–56]. Conversely, Stat5 over-expression increased Lgr5+ cell proliferation and accelerated epithelial regeneration [54–56]. These findings suggest that STAT5 is important in the regulating ISC self-renewal during the inflammatory response.
Summary Thus, during epithelial injury, Lgr5+ stem cells respond to a variety of inflammatory signals to modulate epithelial regeneration. As illustrated in this review, the majority if not all, data on intestinal stem cells responses to injury is focused on Lgr5+ stem cells. Unfortunately, there remains a significant gap in our knowledge of the inflammatory response of other stem cell (i.e. Bmi1+, Lrig1+, Hopx+, among others) populations. Our current understanding of intestinal stem cell responses has been closely linked to our ability to visualize and isolate these stem cells using the described cell markers [11–16]. In this regard, the Lgr5GFP-IRES-CreERT2 mouse first described by Barker et al. (2007) has proven the most robust, and thus made it the easiest to study. This has in large part, been due to the fact that GFP transcription from the Lgr5 locus not only accurately reflects Lgr5 mRNA expression, but also occurs from the same transcript that allows for Cre-mediated lineage tracing [7,57,58,59]. Hence, the cells that express GFP are, for the most part, also the same cells that show stemness, and therefore genetic lineage tracing ability. Unfortunately, this has not always proven to be the case for other established stem cell markers. For instance, FACS isolation of GFP+ cells from Bmi1-expressing cells is only possible from a Bmi1-GFP reporter mouse that was generated using a slightly different genetic strategy than the Bmi1-CreERT2 mouse commonly used for lineage tracing. Moreover, the endogenous Bmi1 gene is ‘‘knocked-out’’ in the Bmi1-GFP reporter mouse, whereas left intact in the Bmi1-CreERT2 mouse [11,60]. Such differences have made it difficult to prove that the cells expressing GFP and CreERT are identical. Furthermore, challenges in our ability to culture and expand non-Lgr5+ stem cell populations have significantly limited our www.sciencedirect.com
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understanding of additional stem cell pools in homeostasis and injury. Future studies of progenitor and/or non-Lgr5+ stem cell pools during epithelial injury will allow discovery of factors that regulate additional stem cell responses. Moreover, as our ability to culture and expand all ISC populations improves, we hope that the therapeutic benefit of ISCs will become a reality.
13. Ramalingam S, Daughtridge GW, Johnston MJ, Gracz AD, Magness ST: Distinct levels of sox9 expression mark colon epithelial stem cells that form colonoids in culture. Am J Physiol Gastrointest Liver Physiol 2012, 302:G10-G20.
Conflict of interest
16. Montgomery RK, Carlone DL, Richmond CA, Farilla L, Kranendonk ME, Henderson DE, Baffour-Awuah NY, Ambruzs DM, Fogli LK, Algra S, Breault DT: Mouse telomerase reverse transcriptase (mtert) expression marks slowly cycling intestinal stem cells. Proc Natl Acad Sci USA 2011, 108:179-184.
Nothing declared.
Acknowledgements Dr. Samuel Asfaha is supported by a CIHR Clinician Scientist Phase I Award and CIHR Operating Grant.
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