Regulation of Intestinal Stem Cells

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1 Regulation of Intestinal Stem Cells Melissa Hirose Wong Department of Dermatology, Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, USA Stem cells are essential for maintaining the tissue integrity of all adult tissues. The manipulation of adult stem cells has the potential for cell regeneration and in curing diseases; however, the intestinal stem cell remains enigmatic. Although much work has focused on characterization of the intestinal stem cell within its in vivo niche, the lack of reliable markers complicates its isolation and therefore its in vitro manipulation. Understanding what regulates the intestinal stem cell within its niche will provide valuable insight into how these cells can be manipulated in culture. Comparing the regulation of this niche in the developing and mature intestine is a valuable untapped resource. A small number of signaling pathways are functionally conserved during development. These pathways are beginning to emerge as critical regulators of the stem cell niche. This review focuses on the regulation of the intestinal stem cell niche. Key words: intestinal stem cell/regulation/small intestine/wnt signaling J Investig Dermatol Symp Proc 9: , 2004 Stem cells hold the promise of cell regeneration and cures for disease; however, the intestinal stem cell remains enigmatic. The lack of reliable markers for the intestinal stem cell complicates its elucidation. In addition, there is disparity between stem cells that reside in (i) the proliferative compartment of the developing intestine, the intervillus region (IVR), and (ii) the crypts of Lieberkühn, the proliferative compartment of the mature intestine. During intestinal morphogenesis, the IVR invaginates to form mature crypts. This physical transformation from a single sheet of cells to a three-dimensional structure alters the relationship between the epithelium and underlying mesenchyme. These changes occur coincidentally with the process of stem cell selection, where a single stem cell in the IVR is selected to become anchored and populate an adult crypt. During formation of crypts, the stem cell pool of the developing proliferative region undergoes a reduction from multiple putative stem cells to a single stem cell per mature crypt. The mechanism that initiates stem cell selection is unknown, but because there is a physical change within epithelial mesenchymal interactions during this period, it is likely that a cell signaling pathway or coordinated signaling pathways are responsible for initiating and facilitating these events. Comparison between the developing intestinal stem cell niche with the mature adult intestinal stem cell niche will likely provide important insight into how the intestinal stem cell is regulated to maintain its proliferative capacity and how a stem cell escapes this regulation to differentiate. The intestine offers a unique model to examine the regulation of stem cells during development and in the adult because both stem cell niches are physically well characterized. In addition, understanding Abbreviations: E, embryonic day; HMG, high mobility group; IVR, intervillus region; LEF, lymphoid enhancing factor; Msi-1, musashi- 1; SHH, sonic hedgehog; TCF, T cell factor the regulation of stem cells within an intestinal context will provide insight into regulation of epithelial turnover and homeostasis in other organ systems such as the skin, lung and mammary gland. The Small Intestinal Functional Unit The mature small intestine is organized into two discrete, yet interactive, functional units, a fully differentiated region and a proliferative region. The differentiated epithelial cells migrate in a linear fashion from the stem cell niche onto the adjacent villi. Since multiple crypts populate a single villus, manipulation of the stem cell population within a crypt allows the descendents to be easily identified. This unique organization facilitates studies that can readily address the regulation of stem cells. The epithelial cells that line the villi, the fingerlike projections protruding into the intestinal lumen, represent the differentiated region. Three differentiated lineages populate the villus epithelium, absorptive enterocytes (which make up 480% of the epithelial population), mucus-producing goblet cells, and enteroendocrine cells (Cheng, 1974; Cheng and Leblond, 1974a c; Paulus et al, 1993). These cells differentiate while undergoing an upward migration from the proliferative zone onto the villus. They migrate in an orderly and linear fashion up from the crypt and onto the adjacent villus where they undergo apoptosis or are sloughed off into the lumen of the gut as they near the villus tip (Schmidt et al, 1985; Hall et al, 1994; Hermiston et al, 1996; Potten, 1997). Because there are differences in villus height and width down the length of the intestine, this process can take approximately 3 5 d. A fourth epithelial lineage, the Paneth cells, which produce anti-microbials and defensins, differentiates as it undergoes a downward migration to reside at the base of the crypt of Lieberkühn (Cheng et al, 1969). The majority of cells within Copyright r 2004 by The Society for Investigative Dermatology, Inc. 224

2 9 : 3 SEPTEMBER 2004 the crypts of Lieberkühn, the flask-shaped mucosal invaginations that line the floor of the small intestine, are undifferentiated epithelial progenitor cells that represent the proliferative region of the mature small intestine. The multipotent intestinal epithelial stem cell has been shown to reside in this zone. Intestinal Stem Cell Hierarchy The physical organization of the small intestinal crypt of Lieberkühn exemplifies an integrated stem cell hierarchy. In the adult mouse, the intestinal epithelial stem cell of the mature small intestine is anchored near the fourth or fifth cell strata at the base of the crypt of Lieberkühn (Loeffler et al, 1993; Potten et al, 1997). This observation is based upon extensive 3 H-thymidine labeling studies to detect labelretaining cells and the position of the lagging edge of labeled, migrating epithelial cells (reviewed in Potten, 1998). By definition, the stem cell can self-renew and give rise to a population of daughter cells that rapidly cycle and differentiate into multiple cell lineages. These cells are called the transient amplifying population and are thought to undergo 4 6 rounds of cell division to amplify the existing pool of stem cell precursors (Winton and Ponder, 1990). Within the crypt, these cells begin to express markers of differentiated cells and become committed along a lineage pathway as they migrate farther from the stem cell source. Therefore, cells closer to the stem cell source are less differentiated than the cells near the crypt opening. Based on the observation that the transient amplifying population is less differentiated when residing near the stem cell(s), Potten and colleagues hypothesize that cells representing the first two rounds of replication are capable of replacing the stem cell population upon stem cell damage and death. Further, they suggest that the stem cell(s) are the most sensitive to cell damage whereas the first two tiers of progenitors in the stem cell hierarchy are relatively resistant to cell damage. This suggestion highlights one of the controversies that exist in intestinal stem cell biology, namely how stem cells are replaced or recruited upon damage. Although there has yet to be definitive proof for existing hypotheses, it is clear that elucidation of this issue will greatly advance our understanding of how the intestinal stem cell is regulated during injury and in the disease state. Intestinal Development and Maturation of the Crypt The exact number of stem cells per mature small intestinal crypt remains an unanswered question. Crypts initially develop from a linear cohort of epithelial cells that reside between villi (IVR). Proliferation in the developing small intestine is confined to the IVR, making it analogous to the proliferative crypt in the mature small intestine. Villi are formed from the pseudostratified gut epithelium around embryonic day(e)14 (Karlsson et al, 2000). The IVR undergoes architectural changes to yield fully formed crypts by the end of the second postnatal week (Calvert and Pothier, 1990). Throughout late fetal life, the IVR contains several active multi-potent stem cells, making it polyclonal in nature INTESTINAL STEM CELLS 225 (Schmidt et al, 1988; Winton and Ponder, 1990; Wong et al, 2000). As the IVR develops into crypts, a poorly understood process of stem cell purification that converts the polyclonal nascent crypt to a monoclonal mature crypt where all epithelial cells share a common genotype occurs. Studies in chimeric mice reveal that the adult small intestinal crypt is derived from a single genotype, suggesting that these crypts originally arose from a single progenitor cell (Schmidt et al, 1988; Wong et al, 2000). This phenomenon has been interpreted to mean that all active stem cells in each purified monoclonal crypt are descendents of a single crypt progenitor cell that occupies the highest position in the established stem cell hierarchy. Although this developmental phenomenon is apparent, it is suggested that mature small intestinal crypts contain several active stem cells (Potten et al, 1995). It is unclear, however, as to exactly how many stem cells populate each crypt. Potten and Hendry suggest a model where there are four to six lineage ancestor cells and up to 30 cells with stem cell potential (first two tiers of the hierarchy). Although detailed cell kinetic and radiobiological studies have provided evidence for this scenario, it remains a conundrum how crypts with multiple stem cells can be derived from a single genotype (Wong et al, 2000). It remains a possibility that crypts are originally populated by a single active stem cell and then, later during development, this population is expanded to accommodate the dynamic turnover of the small intestine. Evidence for regulation of this type of cellular expansion is yet to be identified. Stem Cell Markers for the Intestine The lack of reliable markers for intestinal stem cells has greatly impeded the study of intestinal stem cell biology. Reliable markers would allow for definitive identification of the stem cell population. Furthermore, they would facilitate the ability to isolate and manipulate these cells in vitro, a critical first step for gene therapy approaches. Although previous experimental approaches have predicted stem cell position and numbers (reviewed in Potten, 1998), definitive identification of the intestinal epithelial stem cell remains elusive. Several candidate molecules, however, have been identified primarily based upon their location during intestinal development and in the adult intestine. These molecules include: (i) Tcf-4, a Wnt signaling HMG box transcription factor; (ii) Cdx-1, an intestinal homeobox gene; (iii) Hes-1, a basic helix loop helix, Notch signaling pathway molecule, and most recently; (iv) Musashi-1 (Msi-1), a neural RNA-binding protein (Korinek et al, 1998; Jensen et al, 2000; Lickert et al, 2000; Kayahara et al, 2003; Potten et al, 2003). Although most of these molecules have been shown to participate in maintaining the stem cell niche or in determining lineage allocation, Msi-1 remains an intriguing candidate as a stem cell marker, primarily because its protein localization is consistent with that of intestinal stem cells and it has previously been implicated in maintaining neuronal stem cells (Kayahara et al, 2003; Potten et al, 2003). Using a monoclonal antibody to Msi-1, Potten and colleagues showed high levels of expression in 2-dold neonatal mouse intestine restricted to the developing crypts. In addition, they show that cells interspersed

3 226 WONG JID SYMPOSIUM PROCEEDINGS between the Paneth cells in the mature small intestine are also positive for Msi-1 expression. Although Msi-1 remains an intriguing candidate marker, its expression was observed in both cycling and non-cycling cells, suggesting that this molecule may not be an exclusive stem cell marker. A more global approach for characterizing the intestinal epithelial stem cell is illustrated in a study from Stappenbeck et al (2002). DNA microarrays were utilized to identify genes that are uniquely expressed in a population of small intestinal crypt cells enriched for progenitors. Not surprisingly, this study identified a number of genes involved in maintaining cell cycle, cellular transcription, and translation. The identification of putative markers for the intestinal stem cell, whether by localized expression or by high throughput screening of intestinal mrnas, is a first step toward narrowing the list of potential candidate molecules. To show definitively, however, that the predicted intestinal stem cell markers are indeed markers for stem cells, the ability to isolate and reconstitute lineage allocation in vitro must first be accomplished for the intestinal system. How Are Intestinal Stem Cells Regulated? One of the most intriguing issues in stem cell biology is determining how a stem cell decides to stay anchored within its niche and how it decides to differentiate and migrate. Clearly the cell signaling pathways that are defined through epithelial mesenchymal influences on the stem cell niche are critically important. Interestingly, only a handful of signaling pathways are involved in most of all animal development. These seven pathways include the Hedgehog, Wnt, Tgf-b, Receptor Tyrosine Kinase, Notch, Jak/Stat, and nuclear hormone pathways (reviewed in Pires-daSilva and Sommer, 2003). A number of these signaling pathways are already implicated in stem cell maintenance and it is likely that there will be a temporal and dynamic interaction between these pathways influencing the stem cell niche. Additionally, it is likely that these pathways set up morphogen gradients within the stem cell niche for optimal flexibility and diverse influence of cells within the environment. Wnt signaling The Wnt signaling pathway is one candidate pathway for regulation of stem cell selection during crypt purification. Recently, a Wnt signaling molecule, composed of the required signaling components of b-catenin and the required signaling domains of the downstream HMG-box transcription factor, Lef-1, was overexpressed in the small intestinal epithelium of chimeric transgenic mice (Wong et al, 2002). Overexpression of this Wnt signaling molecule resulted in stimulation of apoptosis in the intestinal stem cells of the IVR as early as E14.5 and throughout the process of crypt purification. Because stem cells overexpressing this fusion molecule were not established within the mature small intestinal crypts, all differentiated cells expressing the fusion molecule were eliminated from the intestine. Only wild-type stem cells were established within the crypts during crypt purification. This suggests that, in a developmental context, putative stem cells receiving high levels of the Wnt signal undergo a negative stem cell selection. The converse experiment, depletion of Wnt signaling using a Tcf-4 knockout mouse was performed (Korinek et al, 1998). This experiment resulted in loss of Tcf-4 expression within the developing stem cell niche, a cessation of cell proliferation and premature differentiation of remaining cells within the proliferative IVR (Korinek et al, 1998). This observation suggests that enterocyte differentiation is a function of the loss of a proliferative Wnt signal. Further, in colorectal cell lines, van de Wetering et al (2002) recently showed that colorectal cancer cells with a disruption in b-catenin signaling rapidly induced G1 arrest resulting in a cell cycle block and subsequent differentiation of the cell line. Because the former experiment was performed by constitutively ablating Tcf-4 and produced an early neonatal lethality, the animals died prior to the complete maturation of the intestine. Therefore, this study does not provide information that may suggest a relationship between Wnt signaling and differentiation of the secretory cell lineages. A temporally regulated gene expression is needed to explore this issue. Taken together these data, however, suggest that the Wnt signaling pathway is critically important for stem cell selection during intestinal development. The evidence presented in this review suggests a model for stem cell selection within the developing intestinal epithelium. The Wnt signaling molecule, a morphogen, has different influences at different concentrations. The Wnt source originates within the underlying mesenchyme (Fig 1). As crypts form, the physical relationship between the overlying, proliferative, linear intervillus epithelium, and the Wnt expressing mesenchyme begin to change. This creates a new relationship between the Wnt secreting cells in the mesenchyme and the overlying stem cells in the epithelium. If the Wnt secreting cells remain localized near the base of the newly formed crypts, a gradient of Wnt morphogen is established where the epithelial cells near the base of the crypt receive high levels of Wnt signaling, the epithelial cells in the central portion of the crypts receive moderate levels of Wnt signaling and the epithelial cells near the top of the crypts receive little or no Wnt signal. The data from the Gordon and Clevers laboratories nicely supports this scenario. We can speculate that the cells near the Wnt source or base of the crypt are influenced by a high level of Wnt signaling, which results in apoptosis and the failure of these cells to be selected as the anchored stem cell during crypt purification. Cells that are far away from the Wnt source see low levels of Wnt signaling resulting in cellular differentiation. Therefore, only stem cells situated at a distinct cell strata will receive the optimal amount of Wnt signaling and will be anchored and maintained in the crypt. Wnt signaling has also been implicated as an important regulator of stem cells in other organ systems. Most recently it been implicated in the ability to expand hematopoietic stem cells in vitro (Reya et al, 2003; Willert et al, 2003), and to respond to Wnt signaling in the nascent environment (reviewed in Eaves, 2003). Reya et al (2003) recently illustrated that overexpression of a stable b-catenin mutant expanded the pool of hematopoietic stem cells in vitro and prolonged their ability to self-renew. Furthermore, addition of purified Wnt3A to cultures recapitulated the proliferative response (Willert et al, 2003). The proliferative response was inhibited by expression of Axin, a known inhibitor of Wnt signaling. In other organ systems, Wnt signaling is critical for progenitor maintenance. Zechner et al

4 9 : 3 SEPTEMBER 2004 INTESTINAL STEM CELLS 227 Figure 1 Model for Wnt signaling participation in selection of intestinal stem cell. The proliferative compartment of the developing intestine is a linear sheet of cells called the intervillus region. Cells in the mesenchyme (dark red) are thought to secrete the Wnt morphogen. During development the intervillus region invaginates to form mature crypts of Lieberkühn. During this process two events occur. First, a physical alteration in the relationship between the epithelium and the underlying mesenchyme, and second, the selection of a single stem cell to populate the crypt. The change in physical conformation changes the relationship of the Wnt secreting cells in the mesenchyme (dark red). Some cells will see high levels of Wnt signaling and undergo apoptosis (dark red arrow). Some cells will see low levels of Wnt signaling and differentiate (light pink arrows). The cell strata that see the optimal amount of Wnt signaling (medium pink arrow) becomes selected and anchored within the stem cell niche. (2003) illustrated that expression of b-catenin can control the size of the neural progenitor pool and influences the decision of this pool to proliferate or differentiate. These data clearly define a role for Wnt signaling in stem cell maintenance and proliferation; however, the challenge will now be to determine how the stem cell remains anchored in its niche despite the influence of Wnt signaling and how it escapes this signal to become a member of the transient amplifying population and proceeds to differentiate. Dynamic interaction between signaling pathways provides a likely scenario for regulating the stem cell s decision to enter the differentiation cascade. The Notch signaling pathway The Notch signaling pathway has been shown to be stimulated by the Wnt signaling pathway (Varnum-Finney et al, 2000). It is unclear what influences a progenitor cell to leave the stem cell niche and adopt a specific fate. Recently the Notch signaling pathway has been shown to play a major role in this decision in the intestine (Yang et al, 2001). Math-1, a Notch signaling component, is a basic helix loop helix transcription factor that has been implicated in cell fate determination in the drosophila nervous system (Ben-Arie et al, 2000; Bermingham et al, 2001). This observation, along with the observation that Math-1 is expressed within the intestinal epithelium, led Yang et al (2001) to wonder if Math-1 had a similar effect on the intestinal stem cell. Analysis of a Math-1 knockout mouse and Math-1 heterozygotes revealed that Math-1 is instrumental in defining the secretory epithelial lineages in the intestine. Math-1 null mice were completely devoid of goblet cells at E18.5. This remarkable finding suggests that Math-1 controls cell fate determination through the Delta Notch signaling pathway. In addition, increased levels of Notch and Delta result in stimulation of Hes-1 expression (Lewis, 1998). Hes-1 is a transcriptional repressor and has recently been suggested that cells expressing Hes-1 maybe precursor cells whereas cells that do not express Hes-1 become committed to different lineages (Jensen et al, 2000; Kayahara et al, 2003). Although the Notch signaling pathway appears to be involved in allowing stem cells to escape the proliferative influence of the Wnt signaling pathway, the balance between Notch signaling and Wnt signaling within the developing IVR and within the mature intestinal crypts has yet to be elucidated. Important temporal expression studies in mice that harbor mutations within these signaling pathways will yield valuable insight into the temporal control of proliferation and differentiation within this niche. Sonic Hedgehog signaling The Wnt and Notch signaling pathways also intersect with the Sonic Hedgehog (Shh) signaling pathway during development. During drosophila wing development, both Wnt and Notch pathways can repress Shh response (Glise et al, 2002), a critical regulatory step in formation of the wing margins. In the intestine, Shh and its homolog Indian Hedgehog are both developmentally expressed in the epithelium, although by E14.5 Shh is restricted to the duodenum (Bitgood and McMahon, 1995). Although it is unclear if Shh or Indian Hedgehog has a role in stem cell maintenance in the intestine, Shh has been shown to influence stem cell division in other systems. Expression of Shh in neuronal stem cells promote their division (Park et al, 2003) and is thought to regulate epithelial progenitors in the lung (Watkins et al, 2003). In addition, expression of Shh is modulated in the hair follicles of mice harboring Wnt mutants (Gat et al, 1998). Prospectus Understanding how intestinal stem cells are regulated in a developmental and mature context hold the potential for manipulating this population of stem cells in tissue regeneration and for gene therapy approaches for disease. Although this field is in its infancy great strides have been made. Signaling pathways such as the Wnt, Notch, and Shh have been identified as key regulators of the stem cell niche. The temporal and spatial patterning of components of these signaling pathways are beginning to be elucidated

5 228 WONG JID SYMPOSIUM PROCEEDINGS (Lickert et al, 2001; Heller et al, 2002; Schroder and Gossler, 2002; McBride et al, 2003). This critical step will lay the groundwork for determining how these signaling pathways are coordinated to control stem cell proliferation and differentiation. With the knowledge of how stem cells are regulated in vivo, we can have the capacity to manipulate the adult stem cell; a feat that holds tremendous implications for improving the quality of life. DOI: /j x Manuscript received July 21, 2003; revised November 17, 2003; accepted for publication December 09, 2003 Address correspondence to: Melissa Hirose Wong, Department of Dermatology, Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon 97239, USA. wongme@ ohsu.edu References Ben-Arie N, Hassan BA, Bermingham NA, et al: Functional conservation of atonal and Math1 in the CNS and PNS. 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