The regulatory niche of intestinal stem cells

Abstract

The niche constitutes a unique category of cells that support the microenvironment for the maintenance and self‐renewal of stem cells. Intestinal stem cells reside at the base of the crypt, which contains adjacent epithelial cells, stromal cells and smooth muscle cells, and soluble and cell‐associated growth and differentiation factors. We summarize here recent advances in our understanding of the crucial role of the niche in regulating stem cells. The stem cell niche maintains a balance among quiescence, proliferation and regeneration of intestinal stem cells after injury. Mesenchymal cells, Paneth cells, immune cells, endothelial cells and neural cells are important regulatory components that secrete niche ligands, growth factors and cytokines. Intestinal homeostasis is regulated by niche signalling pathways, specifically Wnt, bone morphogenetic protein, Notch and epidermal growth factor. These insights into the regulatory stem cell niche during homeostasis and post‐injury regeneration offer the potential to accelerate development of therapies for intestine‐related disorders.

Abbreviations

BMP

bone morphogenetic protein

CBC

crypt base columnar

EGF

epidermal growth factor

ISC

intestinal stem cell

Lgr5

leucine‐rich repeat‐containing G‐protein coupled receptor 5

LRC

label retaining cell

Introduction

The intestinal epithelium, with its rapid turnover, is an excellent system for the study of adult stem cells. It is composed of crypt and villus, and intestinal stem cells (ISCs) are responsible for ongoing epithelial regeneration throughout life. The ISC niche is a complex cellular structure that plays a key role in stem cell maintenance, proliferation and differentiation. The term ‘niche’, coined by Schofield (1978), defines a group of cells playing a crucial role in controlling stem cell fate and maintaining stem cell number by regulating the balance between self‐renewal and differentiation, thus supporting tissue regeneration (Li & Clevers, 2010). Stem cell homeostasis is maintained by mesenchyme and crypt‐based epithelial cells. ISCs reside at the base of crypts, adjacent to Paneth cells, and are surrounded by stromal cells. The niche consists of different cellular components, namely myofibroblasts, endothelial cells, neural cells, lymphocytes, macrophages and smooth muscle cells (Fig. 1). In this review, our focus is mainly on stromal cells, Paneth cells, immune cells, endothelial cells and neural cells. The ISC niche is likely to comprise several different cell types, each of which contributes cell‐associated ligands and chemokines, soluble cytokines and growth factors that regulate stem cell behaviour (Tan & Barker, 2014). To unravel the mysteries of intestinal disease mechanisms and stem cell regeneration, insights into niche components are of high importance. It is known that Wnt, bone morphogenetic protein (BMP), Notch and epidermal growth factor (EGF) signalling pathways are the regulators of stem cell activity, and further studies on these associations with the niche would improve understanding of ISC homeostasis.

Figure 1. Diagram of the regulatory niche of intestinal stem cells .

The stem cell compartment resides at the base of the crypt consisting of CBC and +4 stem cells. Rapidly dividing transit‐amplifying cells arise from these stem cells and differentiate into absorptive lineages (enterocytes) or secretory lineages (enteroendocrine cells, goblet cells, tuft cells and Paneth cells). The niche consists of multiple components and cell types, including extracellular matrix, fibroblasts, myofibroblasts, smooth muscle cells, neural cells, endothelial cells, lymphocytes and macrophages along with secreted factors (Wnt3, EGF), and BMP inhibitors (Noggin, Gremlin and chordin) that support the regulation of stem cell activity. Wnt, BMP, Notch, Hh and EGF signalling pathways are the regulators of stem cell activity (left).

Intestinal stem cells in the niche

Stem cells reside at the base of the crypt and are supported by the microenvironment. Active stem cells are considered to be crypt base columnar (CBC) cells and quiescent stem cells mark the +4 position (Li & Clevers, 2010). CBC cells respond quickly to regenerative niche signals, while +4 label retaining cells (LRCs) remain quiescent during normal homeostasis, but retain the ability to produce other cells after injury (Scoville et al. 2008). Lgr5 (leucine‐rich repeat‐containing G‐protein coupled receptor 5) emerged as a specific and robust marker for CBC cells (Barker et al. 2007). Ascl2, Olfm4, Rnf43, Znrf3, Smoc2, Troy, Prom1, Sox9, and Msi1 were also identified as markers of stem cell populations (Snippert et al. 2009; van der Flier et al. 2009; Hao et al. 2012; Munoz et al. 2012; Fafilek et al. 2013; Schuijers et al. 2014, 2015; Roche et al. 2015). Recently Klf5 (Krüppel‐like factor 5), an additional stem cell marker, was reported in the maintenance of stem cells in the intestinal crypt (Bell & Shroyer, 2015).

The +4 LRCs were discovered by Potten through tritium or Brdu LRC assays (Potten et al. 2002). One of the initial studies showed that phosphatase and tensin homologue (PTEN), especially p‐PTEN, marks +4 stem cells (He et al. 2004; He et al. 2007). The functional validation of +4 stem cells was done by in vivo lineage tracing using Bmi1‐CreER induced mice (Sangiorgi & Capecchi, 2008). Consistent with this observation, Bmi1⁺ cells were characterized as radioresistant and quiescent in contrast to the Lgr5 stem cell population (Yan et al. 2012). mTert expresses at the +4 position identical to LRCs (Breault et al. 2008). Following injury, mTert⁺ cells give rise to all intestinal cell lineages, which was demonstrated by lineage tracing in vivo (Montgomery et al. 2011). A recent study showed that the mTert⁺ dormant stem cell population is regulated by PTEN phosphorylation and nutritional status (Richmond et al. 2015; Sailaja et al. 2015). Takeda et al. demonstrated that Hopx is a +4 stem cell marker and that these Hopx‐expressing cells generate all intestinal epithelial lineages (Takeda et al. 2011 a). Lrig1 is a regulator of the ErbB signalling pathway and is profoundly expressed by ISCs. ISC self‐renewal and proliferation are stimulated by ErbB signalling and controlled by BMP inhibition and Wnt activation (Sato et al. 2009; Wong et al. 2012). In a recent study, Dclk1‐expressing small intestinal epithelial tuft cells, a rare population, were reported to show hallmarks of quiescence with self‐renewal ability (Chandrakesan et al. 2015).

Bmi1, mTert, Hopx and Lrig1 were later reported to be expressed not only in +4 cells, but also in CBC and other progenitor cells (Itzkovitz et al. 2012; Munoz et al. 2012). Some studies reported that +4 cells also express the endocrine marker and that enteroendocrine cells maintain the quiescent stem cell niche (Radford & Lobachevsky, 2006). So, there is considerable debate in the field regarding +4 markers. Recently, similar to the haematopoietic approach, combinatorial surface markers were shown to be able to sort ISCs, and the sorted ISCs could be functionally characterized using an efficient in vitro organoid culture assay (Wang et al. 2013). Characterization and identification of candidate markers must be further validated by gene expression profiling and lineage tracing methods. Broad understanding of intrinsic mechanisms, microenvironmental interactions, and communication with surrounding cells will yield much information about regulatory niche signals during homeostasis.

Differential responses of stem cell subsets in response to injury implicate roles of the niche

The identity of the stem cell is maintained by intrinsic cell and environmental (niche) factors. While substantial efforts have led to progress in uncovering the mechanisms regarding homeostatic ISC maintenance, the regenerative process after ISC injury is still controversial. The deletion of Lgr5 stem cells upon radiation‐caused or diphtheria toxin‐induced ablation does not perturb epithelial homeostasis, indicating the existence of a reserve ISC population (Montgomery et al. 2011; Tian et al. 2011; Takeda et al. 2011 b; Yan et al. 2012). However, following crypt injury, Dll1⁺ secretory progenitor cells are able to reacquire stem cell properties and generate all four secretory cell types, arguing for cellular plasticity as another mechanism (van Es et al. 2012 b). However, the remaining question is what mechanism supports a robust regeneration in response to injury? Recently, keratin‐19 (Krt19) was shown to be expressed at the +4 position, continuing up to the isthmus. These are radiation‐resistant cells that robustly give rise to all intestinal epithelial lineages, including Lgr5 CBCs, in the colon and intestine. Krt19 also marks radioresistant cancer‐initiating cells and regenerates after injury to Lgr5 stem cells (Asfaha et al. 2015).

These studies propose that different subpopulations of stem cells reside in different niches, which provide the required signals to maintain stem cells in different cycling and metabolic states. Hence, the niche regulates stem cells not only in homeostatic conditions, but also in stressed conditions following injury (Scoville et al. 2008). Differential niche regulation may also have a role in gastrointestinal tumours, allowing a cancer stem cell population to be drug‐resistant. Therefore, understanding the different components of the ISC niche following injury and the signals emanating from it is essential in the future.

Stromal microenvironment

The connective tissue cells that support the functioning of parenchymal cells in an organ are called stromal cells. These are also often referred to as non‐haematopoietic, non‐epithelial and non‐endothelial in origin (Owens, 2015). Stromal cells and subepithelial myofibroblasts exist in the lamina propria, located beneath the intestinal crypts, which supports ISCs during intestinal morphogenesis, differentiation and proliferation (McLin et al. 2009; Mifflin et al. 2011). Intestinal subepithelial myofibroblasts exhibit qualities of smooth muscle cells and fibroblasts (Pinchuk et al. 2010; Powell et al. 2011). Myofibroblasts maintain and support stem cells and lead to expansion of intestinal epithelium. The linkage among crypt stem cells, Paneth cells and myofibroblasts requires investigation to further delineate their molecular interactions (Powell et al. 2011). Farin et al. revealed that stromal cells support the formation of intestinal epithelium by the Wnt signalling pathway (Farin et al. 2012). A recent study showed that epithelial Wnt is non‐essential, and stromal cells endogenously express Rspo3, a secreted Wnt agonist (an R‐spondin family member), which supports regeneration of intestinal epithelium (Kabiri et al. 2014). Hedgehog (Hh) signalling, an important component of epithelial–mesenchymal cross‐talk, also contributes to establishing epithelial stem cells in the niche by inducing stromal BMP synthesis (Vries et al. 2010). Hh signalling within the stroma results in decreased epithelial proliferation and expansion of smooth muscle cells and myofibroblasts. A similar phenotype was observed in mice overexpressing Noggin (a BMP inhibitor) indicating the importance of BMPs as downstream mediators of Hh signalling (Vries et al. 2010; Zacharias et al. 2010). Snai1, which also regulates the epithelial‐to‐mesenchymal transition, is reported to be necessary for maintenance of CBC stem cells (Horvay et al. 2011).

Stromal cells are the mesenchymal elements that have also been implicated as key players in regenerating injured tissue following trauma. These mesenchymal cells possess similar marker molecules, origins and coordinated biological functions and provide a microenvironment for ISC maintenance (He et al. 2004; Pinchuk et al. 2010). The isolation and characterization of mesenchymal niche cells are critical in determining their functional regulation of the ISC niche. Furthermore, the mechanisms that regulate these interactions between mesenchymal and crypt base epithelial cells remain unclear. A better knowledge of stromal cells in the niche is indispensable for understanding the mechanisms of homeostasis and disease. During inflammation, tissue stromal cells experience immunological changes (Pinchuk et al. 2010). Since the effects of these stromal cells are poorly understood and advances in the field are minimal, further studies on stromal cell functions and the ISC niche may lead to next‐generation cell‐based therapies for intestinal bowel disorders.

Immune, endothelial and neural cells in the niche

In the small intestine, endothelial cells and neural cells are present along with connective tissue fibroblasts. The study by Bjerknes and Cheng introduced enteric neurons and blood vessels as niche cells surrounding the intestinal crypt (Bjerknes & Cheng, 2001; Mills & Gordon, 2001). Neural cells play a key role in regulating epithelial growth. Glucagon‐like peptide 2 (GLP‐2) produced by eneteroendocrine cells signals to underlying enteric neurons that express the GLP‐2 receptor, which stimulates the proliferation of enterocytes (Bjerknes & Cheng, 2001). Endothelial cells were identified as key players of the niche as there was no epithelial cell loss when endothelial apoptosis was blocked by using basic fibroblast growth factor (bFGF) after radiation damage (Bjerknes & Cheng, 2001; Paris et al. 2001). Immune cells contribute to the protection of epithelial surfaces by releasing factors and promoting tissue repair. Both epithelial cells and dendritic cells interact to maintain immune balance in small intestine (Rimoldi et al. 2005). Regulatory T cells, which have the capacity to modulate the immune system, along with other mesenchymal cells, macrophages and endothelial cells, also play an important role in maintaining the regulatory niche (Paris et al. 2001; Akcora et al. 2013). Regulatory T cells, a subset of CD4⁺ T cells, are central players for maintaining intestinal homeostasis (Korn et al. 2014). Colony stimulating factor (CSF1) receptor, which is expressed on macrophages, fashions the ISC niche (Akcora et al. 2013). Growth factors, cytokines and ligands are secreted by intestinal immune cells and stromal cells that regulate ISCs (Pinchuk et al. 2010). Stromal cells respond to the immune cell‐derived cytokines interleukin (IL)‐1α and IL‐1β, with relevance to intestinal inflammatory diseases (Okuno et al. 2002). The crypt is also influenced by external signals, such as IL‐22 produced by innate lymphoid cells, that maintain epithelial integrity and protect stem cells against damage (Hanash et al. 2012). Hence, it is critically important to investigate the role of endothelial, neural and immune cells as niche cells in the intestine, as their functional properties are least known.

Paneth cells as a regulatory niche

Paneth cells are the cells identified by Joseph Paneth as columnar epithelial cells of the secretory lineage with cytoplasm filled with large granules and located at the base of the crypt (Clevers, 2013). CBC cells are interdigitated between Paneth cells, and the function of Paneth cells involves production of growth factors such as Wnt, Notch and EGF as niche signals to CBC stem cells. Yilmaz et al. proposed that Paneth cells, a vital member of the ISC niche, augment stem cell function in response to fasting. Refeeding starved mice regulates mTORC1 in Paneth cells. These results establish that mTORC1 regulates self‐renewal of the ISC niche and emphasize its significance in supporting the functionality of stem cells (Yilmaz et al. 2012).

In intestinal crypts, Paneth cells constitute the niche for stem cells. The communication between CBC cells and Paneth cells is seen in in vitro intestinal organoid culture. Recent research revealed that co‐culturing ISCs with Paneth cells or exogenous Wnt3a improves growth efficiency of Lgr5 stem cells (Sato et al. 2011). As colon is devoid of Paneth cells, CD24⁺ cells and C‐Kit⁺ colonic goblet cells located adjacent to the Lgr5⁺ stem cells at the base of crypt have been proposed to be niche components (Rothenberg et al. 2012). There is controversy about the role of Paneth cells as a niche since ablation of Paneth cells did not affect number and function of Lgr5⁺ ISCs (Durand et al. 2012; Kim et al. 2012). Interestingly, another report showed that deletion of Wnt3 had no effect on stem cell function in adult mice, although Wnt is necessary for organoid cultures (Farin et al. 2012). However, Paneth cells play a role in facilitating ISC recovery post‐intestinal injury (Parry et al. 2013). Taken together, Paneth cells provide several niche factors in vivo, and there is also redundancy when compared with stromal cells.

Regulatory signalling pathways in the niche

Several regulatory signalling pathways are known for their importance in the niche. The Wnt pathway is considered to be the crucial pathway for maintaining self‐renewal and proliferation of ISCs. Wnt signals are exhibited more along the base of the crypt and less towards the villus (Vries et al. 2010). Knock‐out of Tcf, Dkk1 (a secreted Wnt antagonist), Ctnnb1 and c‐Myc (a Wnt target gene) greatly affects the intestine's proliferative compartments, indicating the importance of Wnt signals in forming stem cell compartments (Kuhnert et al. 2004; Muncan et al. 2006; Fevr et al. 2007; van Es et al. 2012 a) . The WNT agonist roof plate‐specific spondin 1 (R‐spondin) and knock‐out of the APC gene readily drive hyperplasia in intestine and colon (Kim et al. 2005). Simultaneous deletion of Rnf43 (ring finger protein 43) and Znrf3 (zinc and ring finger 3), which are Wnt target genes, is key to modulation of the Wnt signal and drives hyperplasia in intestine (Hao et al. 2012). Wnt ligands are secreted by pericryptal stromal cells and epithelial cells, including Paneth cells that produce Wnt3 (Sato et al. 2011). Even without Wnt3, Wnt2b can support the growth of enteroids (Farin et al. 2012), and Lgr receptors mediate R‐spondin signals in enhancing the Wnt signalling pathway (de Lau et al. 2011). Although numerous studies show that Wnt is critical for stem cell function, other studies question the need for secreted Wnt and its source in vivo (San Roman et al. 2014). Very recent findings reveal Fzd7 is highly expressed in Lgr5 stem cells and mediates Wnt signalling (Flanagan et al. 2015).

Mesenchymal derived BMPs belong to the transforming growth factor β (TGFβ) family. In contrast to Wnt signalling, BMP signals are exhibited high at the villus and less towards the base of the crypt, inhibiting stem cell renewal and supporting epithelial differentiation (He et al. 2004). Mesenchymal cells express BMP4, including cells that are adjacent to ISCs. In the submucosal region, the BMP inhibitor Noggin was expressed predominantly adjacent to the crypt bottom and was occasionally detected only in ISCs, thus supporting CBC cell proliferation via inhibiting BMP (He et al. 2004). In addition, gremlin 1, gremlin 2 and chordin are also highly expressed in the submucosa to suppress BMP signalling (He et al. 2004; Pinchuk et al. 2010).

Notch is another regulator of the niche that is required for ISC maintenance, and furthermore, it requires cell–cell interactions, suggesting the stem cell is regulated by adjacent epithelial cells and not stromal cells to deliver the Notch signal (Pellegrinet et al. 2011; VanDussen et al. 2012; Tian et al. 2015). Knock‐out of RBPjκ, simultaneous inactivation of Dll1 and Dll4 (membrane bound notch ligands), or double deletion of Notch1 and ‐2 impairs secretory lineage and cellular proliferation (van Es et al. 2005; Riccio et al. 2008; Pellegrinet et al. 2011). Further understanding of how the various signalling pathways are integrated to regulate ISC function is a major challenge in the field. A recent study presented data suggesting Notch and Wnt pathway interactions regulate ISCs (Tian et al. 2015). EGF‐like growth factors also regulate stem cell activities through different signalling pathways such as phosphoinositide 3‐kinase (PI3K), protein kinase B (Akt), Ras, Raf, mitogen‐activated protein kinase kinase (MEK), mitogen‐activated protein kinase (MAPK) and protein kinase C (PKC) (Normanno et al. 2006). Hence, there is a need to further understand how the various niche factors are integrated to maintain homeostasis as well as to respond to challenges that modulate the ISC compartment.

Understanding signals emanating from the niche provided researchers with the critical insight to add different growth factors such as EGF, R‐spondin‐1 and Noggin to in vitro organoid culture (Fig. 2) (Sato et al. 2009). Interestingly, enteroids derived from mouse crypts display spontaneous oscillations of gene expression (Moore et al. 2014), suggesting the presence of a clock mechanism inherent to the intestinal epithelium. Of practical consequence is the observation that these rhythms appear to influence the responses to growth factors. The organoids grown in culture with niche factors self‐renew and produce all types of epithelial cells, resembling the intestinal epithelium and mimicking the stem cell niche in vivo, and hence can be used instead of cell lines (van de Wetering et al. 2015). Organoid culture may also represent a cheap and robust alternative to xenograft‐based drug studies. Hence, these organoids are useful in cancer genetics to allow design of personalized therapy.

Figure 2. Ex vivo organoid culture .

Stem cells (Lgr5⁺) plated into laminin‐rich matrigel supplemented with a cocktail of niche factors including Noggin, R‐spondin 1 and EGF generate self‐renewing epithelial organoids resembling the stem cell niche in vivo.

Summary and perspectives

The signals emanating from the niche in epithelial cells and stromal cells regulate intestinal homeostasis. Wnt signals are indispensable for ISC self‐renewal and the differentiation of Paneth cells, BMP signals for balancing ISC maintenance and activation, and Notch signals for specifying the absorptive and secretory lineage crucial to establishing the niche during homeostatic and injury conditions. The ISC niche maintains stem cell number. Although there has been much progress over the last few years in identifying which important cellular components regulate the ISC niche, many unresolved issues remain. The identity of ‘+4 stem cells’ and how they respond to multiple niche signals during homeostasis, and particularly in response to injury, remain largely unknown. The origin of stromal cells and their functional identity are largely unclear. Identification of proper markers to define immune cells, endothelial cells and neural cells in regulating ISCs will help us understand niche interactions in homeostatic and stressed or injury conditions. There are certain enduring questions in understanding the signalling mechanisms which regulate the niche. Uncovering the role of Wnt, Hh, BMP, Notch and EGF signalling in stromal cells will help illuminate these questions. Further research on the function of stromal cells in response to the immune system with a variety of cytokines will enlighten the field and may lead to new therapies in treating gastrointestinal disorders. In the future, artificial ex vivo niches may provide intriguing opportunities for regeneration‐based treatment.

Additional information

Competing interests

None of the authors has any conflicts of interests.

Author contributions

All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was funded by NIDDK (U01DK085507).

Biographies

Badi Sri Sailaja is a postdoctoral fellow at Stowers Institute for Medical Research, working on characterization of intestinal stem cells and their niche. She received her PhD from Hebrew University of Jerusalem, Israel.

Xi He is a senior research specialist at Stowers, investigating intestinal and haematopoietic stem cells. She has participated in numerous studies and published over 30 papers.

Linheng Li is an investigator at Stowers, best known for combining genomics and genetics to study haematopoietic and intestinal stem cells and their associated niches. He was among the first to identify the endosteal niche and among the first to propose a model of co‐existing quiescent and active adult stem cells in the same tissue in mammals. He received his PhD from New York University Medical Center and has contributed to the field by characterizing and demonstrating the roles of several developmental signalling pathways in regulation of stem cells.