The maintenance of epithelium homeostasis in the adult small intestine

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The maintenance of epithelium homeostasis in the adult small intestine

Homeostasis of the intestinal epithelium is ensured by the continuous cells division in the crypt and apoptosis both in the crypt and at the top of the villi. Maintenance of stem cell, cell migration and apoptosis are precisely orchestrated by biochemical and mechanical signaling. As previously discussed in embryonic morphogenesis of the intestine, those signals have various origins: homotypic and heterotypic signaling, cell matrix interaction and long range interactions with regards to the underlying tissues like muscular layers. The following sections details how these actors support intestinal homeostasis. With respect to the aim of tissue engineering which is to recreate a microenvironment that induces the differentiation of cells into a specific tissue, evaluating the influence of each parameters is essential in order to simulate them in vitro.

Stem cells and epithelium renewal

The following section introduces the specific location of stem cells in the crypt, how they self-renew and generate their progeny. The factors responsible for their maintenance and the induction of a specific fate to their progeny are also presented. The spatial restriction of these factors to the low part of the crypt confirms the vision of the crypt as a stem cell niche.

Stem cell hierarchy and crypt plasticity

In 1974, crypt base columnar (CBC) cells localized at the bottom of the crypt were first identified as stem cells by Cheng & Leblond (Cheng and Leblond 1974). They injected tritiated thymidin in mice to determine where the dividing cells were located. Unfortunately the-radiation of the tritium killed the CBC cells undergoing mitosis during the injection in the crypt. Apoptotic CBC cells were phagocytosed by their neighboring CBC cells thus exhibiting a large radiolabeled phagosome. After 12 hours, phagosomes were founded in the mid crypt and after 30 hours they appeared in the enterocytes and Paneth cells. Since phagosomes were first identified in the CBC cells and then in differentiated cell types, they concluded that CBCs behave as the stem cells of the epithelium as they are multipotent and have the ability to self-renew (Cheng and Leblond 1974). More recently, the location of CBC stem cells was precisely determined in the study of Barker et al (Barker et al. 2007), who identified Lgr5 as a marker of those cells. They located cells expressing Lgr5 (Lgr5+ cells) at the bottom of the crypt intercalated between Paneth cells and counted approximatively 3-4 Lgr5+ cells per crypt.
They then used Cre recombinase to follow their progeny. Cre-recombinase catalyzes recombination across DNA sequences called lox P. Cre recombinase can be associated to estrogen receptor (ERT) to permit temporal activation of the cre-recombinase. When Cre recombinase is bound to estrogen receptor it cannot enter the nucleus and therefore is not active. In presence of tamoxifen, tamoxifen binds to the ERT receptor and induces a change in its conformation that allows the cre-ERT construct to enter the nucleus and induce targeted mutation namely the fusion of two lox P sequences and the excision of the intermediate sequence. (Figure 7).
Figure 7: Cre recombinase technique in cells expressing Lgr5. In absence of tamoxifen Cre recombinase bound to ERT is restricted to the cytoplasma and Lac Z is not expressed. Following the addition of tamoxifen, Cre recombinase translocates to the nucleus, recombines loxp sequences resulting in the expression of LacZ In this study, the gene coding for the enzyme Cre-recombinase-ERT was located in the first exon of the gene coding for Lgr5. As a result, Cre recombinase-ERT was only present in the cytoplasm of cells expressing Lgr5. In the nucleus two lox P sequences flanked a silence sequence. When Cre recombinase was absent this silence sequence was transcribed. In the presence of Cre recombinase the two lox P were fused, the silence sequence was excised and the Lac Z gene following the second lox P sequence was transcribed (Figure 7). Therefore following tamoxifen injection, Lac Z was expressed only in the Lgr5+ cells. After 5 days, they observed ribbons of cells expressing Lac Z starting from the bottom of the crypt and reaching the top of the villus. It confirmed that the progeny of Lgr5+ cells can differentiate into the 6 differentiated cell types. After 60 days cells expressing Lac Z were still found testifying of the ability of Lgr5+ to self-renew (Figure 8).
Figure 8: Lineage tracing in the small intestine. Analysis of Lac Z expression (blue cells) c)1 day after tamoxifen induction, d) 5 days after injection and e) 60 days after injection. Imported from (Barker et al. 2007).
In 1977, Potten suspected the existence of another stem cell subpopulation located at the +4 location (fourth position starting from the crypt base) in the crypt as those cells were also sensible to irradiation (C S Potten 1977). Since those cells were also able to retain labeled DNA over cell cycles (C S Potten, Kovacs, and Hamilton 1974), it was suggested that these +4 cells could be stem cells. Indeed the fact that these cells retain labeled DNA indicates that they divide asymmetrically: DNA template strand is preserved in the stem cell while newly synthesized strands with potential errors are sent to the daughter cell (C S Potten 1977). In addition, their sensitivity to radiation resembles the behavior of stem cells that undergo apoptosis in case of damage to avoid the apparition of errors in their template DNA strands (Christopher S Potten, Owen, and Booth 2002). The study of Sangiorgy et al (Sangiorgi and Capecchi 2008) confirmed that +4 cells can be considered as intestinal stem cells since after 12 months, their lineage was still present testifying of their ability to self-renew and the five differentiated cell types were found in their progeny. In their experiments, they defined Bmi1 as a marker of those +4 and used the Cre recombinase approach to follow the cells expressing Bmi1 and their progeny. They also noticed that +4 stem cells were slow cycling cells; this observation could explain their ability to retain DNA. Since some crypts did not expressed Bmi1 stem cells but where still active, they confirmed the existence of the additional Lgr5 stem cell subpopulation.
Figure 9: Summary of the characteristics and the markers of the two subpopulations of stem cells: the crypt base columnar (CBC/ Lgr5+) stem cells and +4 stem cells. Imported from (Barker 2014) with modifications.
The existence of two subpopulations of stem cells in the crypt is now globally admitted and provides a more plastic model of the origin of stem cells (Figure 9). The Lgr5+ / CBC cells are considered as the active stem cells responsible for daily epithelial homeostasis while +4 stem cell population represents a more quiescent/ slow cycling population that is activated following tissue injury (Barker 2014; Gracz and Magness 2014; C S Potten 1977; Sangiorgi and Capecchi 2008). In case of major injury TA cells (Lgr5-) are able to dedifferentiate and to adopt the stem cell fate (Lgr5+) (Barker 2014; Gracz and Magness 2014). Such observations suggest some hierarchy among the stem cells present in the crypts (Barker 2014; Christopher S Potten, Owen, and Booth 2002). The plasticity of the cell populations in the crypt might be sustained by the specific signaling and microenvironment occurring in the stem cell niche (Barker 2014).

Asymmetrical stem cell division vs neutral drift in the crypt

Despite the permanent dividing stem cells, intestinal tissue homeostasis imposes the total number of cells to remain constant. Therefore it implies some asymmetry in the fate of daughter cells, half of them remaining stem cells. This asymmetry can be assessed at the scale of individual stem cell (division asymmetry) or of the whole crypt population (population asymmetry) (Lopez-Garcia et al. 2010). In population asymmetry, the loss of one stem cell dividing into two TA daughter cells is compensated by the division of another stem cell into two daughter stem cells.
Potten (C S Potten 1977) first made the assumption that asymmetric division was the rule regarding individual stem cell division. Template DNA strands segregates from newly synthesized strands and remains in the stem cells to avoid the emergence of any error in the stem cell DNA. This hypothesis was experimentally confirmed by Quyn et al (Quyn et al. 2010) study. They labeled the newly synthesized DNA with EdU in the crypt and observed that for the cell located at position 4 and below, the mitotic spindle of dividing cells was oriented perpendicular to the surface and that in most cases division was asymmetric. (Figure 10).
Figure 10: Asymmetric distribution of DNA in dividing stem cells 1 day after EdU injection. Newly synthesized DNA EdU labeled (in green) and template DNA labeled with Dapi (blue). Imported from (Quyn et al. 2010).
However controversial studies are in favor of an asymmetry at the population level. A first observation was performed on chimaeric mice which are mice that are constituted of two populations of genetically distinct cells (Schmidt, Winton, and Ponder 1988). Two days after birth, the crypts that were at the borders between two chimaeric patches expressed cells of each population indicating the existence of stem cells of both populations (polyclonality). Two weeks after, mixed crypts tended to express one cell population more than the other with a gradual disappearance of mixed crypts. At the adult stage all crypts were only formed with one population of cells indicating the prevalence of one stem cell type in each crypt (monoclonality). In conclusion, the crypts that were initially polyclonal shift to monoclonality over time (Schmidt, Winton, and Ponder 1988). This phenomenon called neutral drift was also observed in adult mice. Snippert et al (Snippert et al. 2010), generated crypts with stem cells with random colors in a 10 weeks old mutant mice using the Cre recombinase approach previously presented. The genetic recombination generated stem cells that expressed either green nucleus, blue membrane, or yellow or red cytoplasm in presence of tamoxifen. The injection of tamoxifen thus genetically modifies randomly the Lgr5+ cells resulting in stem cell clones with different colors in the crypt (polyclonal crypt). In the following weeks the authors observed the prevalence of one color per crypt (Figure 10). After 8 weeks almost all crypts were single colored indicating that they drift to monoclonality through time. The villi are however polyclonal since their cellular turn-over is fueled by several surrounding crypts (Figure 11). One explanation for this neutral drift is that Lgr5+ stem cells rather divide symmetrically generating two potential Lgr5+ stem cells, then local signaling from the niche and competition for available niche space determines the fate of the daughter cells. Thus the asymmetry that guarantees the homeostasis of the tissue is noticed at the population level and would be the result of neutral competition between equal stem cells in the crypt (Snippert et al. 2010).
Figure 11: Neutral drift from polyclonality to monoclonality through time in crypt. a) lineage tracing at the bottom of the crypts in the x-z plane polyclonal crypts are marked with a white dashed circle. b) Transversal cut of polyclonal villi and surrounding monoclonal crypts in the intestine 8 weeks after tamoxifen injection.
Regarding these controversial studies, it is hard to conclude weather asymmetrical or symmetrical stem cell division is prevalent in the crypt. It remains however certain that some stem cells are maintained in the crypt since several studies could follow the lineage of single stem cells during months (Barker et al. 2007; Cheng and Leblond 1974; Sangiorgi and Capecchi 2008; Snippert et al. 2010). In order to maintain stemness at the bottom of the crypt, two major signaling pathways, Wnt and the Notch, were identified as crucial.
Wnt ligands are known to be secreted by both Paneth cells and myofibroblasts underlying the epithelial layer. A recent study suggests the presence of an additional source of Wnt since the integrity of the crypts were maintained even though signal emanating from both Paneth and myofibroblasts were inhibited (San Roman et al. 2014). Wnt ligand binds to the membrane receptor and induces the inactivation of the β catenin degradation pathway. β catenin then accumulates in the cytoplasm, translocates to the nucleus and binds to the promoters of the transcription complex of Wnt target genes (van der Flier and Clevers 2009). The amount of Wnt in the crypt is tightly regulated through a feedback loop from Wnt target genes. As an example among the genes that are express upon Wnt activation, Lgr5 gene amplifies Wnt signal whereas Rhf43 and Troy down regulate it (Barker 2014). Since Wnt sources are mostly located at the bottom of the crypt, Wnt diffuses as a gradient along the crypt axis (M. H. Wong 2004). Therefore the stemness of stem cells is maintain as they are under the direct influence of Wnt but as cells migrate upwards in the crypt they receive low level of Wnt and therefore are engaged in the differentiation process (M. H. Wong 2004). In this respect, Sansom et al (Sansom et al. 2004) reported that an overexpression of Wnt strongly impedes differentiation by reducing the number of differentiated cells and increasing the number of transit amplifying cells.
Wnt also participates in the segregation between stem cells and differentiating cells as it upregulates the expression of EphB2 and EphB3 receptors and down regulate the expression of ephrin b1 ligand ( see § 1.1.b for the mechanism of Eph/ephrin interaction). Therefore, a decreasing gradient of EphB2 receptor is observed from the bottom to the top of the crypt in direct correlation with Wnt gradient, while ephrin b1 gradient follows the opposite tendency (Figure 12). The repulsive forces generated between cells carrying ephrin ligand and those carrying Eph receptors participate in cell segregation and could promote the upward migration of differentiating cells. Similarly, the expression EphB3 receptors at the surface of Paneth cells could be responsible for their downward migration (Batlle et al. 2002).
Figure 12: Wnt induce spatial segregation between proliferative and differentiated cells by upregulating the expression of EphB 2 and down regulating the expression of ephrins. Adapted from (Peifer 2002).
Wnt is also involved in the differentiation process as it drives the maturation of Paneth cells and promotes the differentiation of secretory lineages. Pinto et al (Pinto et al. 2003) observed that the inhibition of the Wnt pathway in mice mutant secreting Dickkopf 1 (Wnt inhibitor) correlates with the loss of crypts and the lack of secretory lineage although absorptive lineage did not seem affected. Wnt would thus have longer range of action than expected and can confer TA cells the potentiality to differentiate into secretory lineage.
Notch signaling is also involved in stem cells maintenance. Notch is a juxtacrine signaling. Therefore, Notch ligands and receptors are expressed at the membrane surface epithelial cells and physical contact between two adjacent cells is needed for this pathway to be activated. Notch expression is restricted to intestinal crypts (van der Flier and Clevers 2009). Inhibition of Notch signaling leads to a conversion of all cells to the secretory lineage, even in the crypt where proliferation is stopped. The overexpression of Wnt in those crypts is not sufficient to overcome this stemness loss, indicating that both Wnt and Notch signaling are required for stem cell maintenance (Crosnier, Stamataki, and Lewis 2006). When Notch is overexpressed, the number of secretory cells is reduced and most of the cells differentiated into enterocytes. Therefore Notch signaling is considered to balance between the differentiation into secretory versus absorptive lineage (Crosnier, Stamataki, and Lewis 2006; van der Flier and Clevers 2009).
Wnt and Notch pathways both play key roles in the maintenance of the stem cells in the crypt (Figure 13). In this regards their respective concentration in crypts has to be optimal to ensure the homeostasis of the intestinal tissue. Homeostasis of the intestinal tissue is ensured by a constant division of stem cells in the crypt and apoptosis of differentiated cells at the villus tip but, in the meantime, it implies that epithelial cell directionally migrate from crypt to villus.
As evidenced by the studies that follow the evolution of labeled stem cell progeny (Sangiorgi and Capecchi 2008; Snippert et al. 2010), epithelial cells in the intestine migrate upwards from the basal part of the crypt to the tip of the villi in approximately 5 days. Stem cells are hypothesized to drive cell migration as they exert a mitotic pressure by dividing at the bottom of the crypt (Meineke, Potten, and Loeffler 2001). The directionality of this pressure driven passive movement is set by dividing cells at the bottom of the crypt and the gap left by apoptotic cells at the top of the villus. However, Frey et al (Frey, Golovin, and Polk 2004) noted that the ability for cell to migrate depends on their gene expression. In their experiment, they noticed that EGF (Epidermal Growth Factor) simulated migration in epithelial cell expressing active P38 protein (member of MAPK signaling family) whereas it induced the proliferation of cells with inactive P38. This finding suggests that migration can be induced by external factors but only in the cells expressing the right proteome. Other studies suggest that cell migration is more an active than passive driven phenomenon. Kaur et Potten (Kaur and Potten 1986a) actually observed that epithelial cells were still migrating even though cell division erased by either irradiation or drugs administration. Migrating cells normally express motility proteins and exhibit a motile phenotype. Even though the presence of lamellipodia on the basal part of epithelial cells has still not been observed in vivo, Kaur et Potten noticed that the inhibition of protein synthesis in vivo induce an inhibition of cell migration in the crypt but not in the villi. Therefore they deduced that cell migration in the crypt was achieved through motility protein synthesis. On the contrary, cell migration in the villi was disrupted when noradrenaline, a drug that blocks smooth muscle contraction, was administered to the mice. They thus assumed that smooth muscle contraction stimulates cell migration in the villi. The distinct mechanisms driving cell migration in the crypt or in the villi might explain the fact that migration velocities measured in the villi and in the crypt are not correlated in time driving (Kaur and Potten 1986b). Subepithelial fibroblasts underneath the BM might also be involved in epithelial cell migration.

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Subepithelial fibroblasts and epithelium homeostasis

Subepithelial fibroblasts are essential to the balance of the intestine under various aspects. Indeed, they secrete constituent of the ECM and of the basement membrane, act as mechanical support of the villi structure, induce both maintenance epithelial stemness and differentiation through paracrine signaling, and are possibly induce epithelial cell migration.
Subepithelial fibroblasts are present just below the basement membrane in the lamina propria (Figure 14 a). At the lower part of the crypt, they exhibit a spindle shape and are organized in two to three overlapping layers surrounding the bottom of the crypt. In this portion of the crypt, the fibroblasts are so densely packed that they are the only mesenchymal cells in contact with the BM (Marsh and Trier 1974a; Parker, Barnes, and Kaye 1974). Their sheet-like processes, beneath the BM, cover an area of approximately height adjacent epithelial cells. In the upper part of the crypt and proximal portions of the villi, the density of fibroblast decreases as they are organized in a single discontinuous layer. This discontinuous network allows other mesenchymal cell types (plasma cells, endothelial cells, leukocytes or macrophages) to be in direct contact with the BM (Marsh and Trier 1974a). From the basal part to the top of the villi, network of fibroblasts gets even sparser. The fibroblasts are more stellate and they interact with the BM through finger-like processes (Marsh and Trier 1974a). In the villi, fibroblasts processes together with collagen fibrils form a reticular sheet with numerous pores ranging from 3 to 7µm called foramina (Furuya and Furuya 2007).
Figure 14: Cellular network of intestinal subepithelial fibroblasts. (a) Scanning electron micrograph of subepithelial fibroblasts (*) connected as a syncitium underneath columnar epithelial cells (EP) in rat villi. Arrows indicate apical surface goblet. Scale bar 20µm. Imported from (Desaki, Fujiwara, and Komuro 1984).(b) Schematic representation of subepithelial fibroblasts along the crypt villus axis. Spindle shaped fibroblasts proliferate in the crypt and then differentiate as they migrate along the crypt-villus axis. While they migrate they switch from spindle shape to stellate shape, their level of expression of αSMA decreases but level of desmin increases. Imported from (Furuya and Furuya 2007).
The first layer of pericryptal fibroblasts were identified as myofibroblasts due to their contractile apparatus made of α smooth muscle actin (αSMA) and non-muscle myosin (Mifflin et al. 2011). They are attached to the ECM through specialized fibronexus adhesion complex connecting actin bundles to extracellular fibronectin in a plaque-like structure. Fibronexus are particularly efficient to mechano transduce the external ECM tension to the stress fibers but also to transmit cell contraction to the external matrix (Hinz 2006; Mifflin et al. 2011; Tomasek et al. 2002). Spindle shaped myofibroblast contains high level of αSMA and low level of desmin. On the contrary, stellate fibroblasts in the villi exhibit a high level of desmin but low αSMA (Furuya and Furuya 2007) (Figure 14 b). As desmin happens to be a marker of fibroblast differentiation and also because the organelles, Golgi and granular apparatus were absent or immature in pericryptal fibroblasts, fibroblasts in the villi were considered as more mature than pericryptal fibroblasts (Furuya and Furuya 2007; Parker, Barnes, and Kaye 1974).
Marsh et al (Marsh and Trier 1974b) observed that subepithelial fibroblasts were able to divide in the low crypt region and to migrate upwards to the distal villi part. Tritiated thymidine injection in mice revealed that subepithelial fibroblasts were dividing slower than epithelial stem cells. However, fibroblasts seemed to precede epithelial cells during upward migration. Taken together these observations suggest that subepithelial fibroblasts differentiate while they migrate along the crypt villus axis but also that their migration could initiate epithelial cell migration. It is however important to notice that other studies failed to prove the existence of this co-migration (Neal and Potten 1981).
All epithelial fibroblasts (myofibroblasts and fibroblasts) are connected through gap junction (Furuya and Furuya 2007; Mifflin et al. 2011). Gap junctions are made of connexins assembled in a tubular transmembrane structure that ensures direct communication between two adjacent cell cytoplasma. Information is thus rapidly transmitted to the whole network of fibroblasts. It is particularly efficient in case of injury: the damaged epithelium secretes ATP which is transduced in subepithelial fibroblasts and results in fibroblast contraction due to a sudden increase of calcium level. Calcium wave propagates to other fibroblasts via gap junction provoking the contraction of the whole fibroblast network (Furuya and Furuya 2007). Fibroblasts contraction reduces the size of the sieving of the collagen/ fibroblast network and therefore temporary modifies the mechanical properties of the villi and also its absorptive properties. Other substances like substance P neurotransmitter secreted by neurons from lamina propria or endothelin from endothelial cells also trigger the increase of Ca2+ intracellular level in subepithelial fibroblasts (Furuya and Furuya 2013). In consequence subepithelial fibroblast network is necessary to integrate external signals and to provide the villi the adequate mechanical properties in response to these stimuli (Furuya and Furuya 2007).
Subepithelial fibroblasts are also involved in the maintenance of differentiated/ proliferative cells segregation through Bmp signaling pathway. They secrete Bmp4 ligand that binds type I and type II serine/ threonine kinase receptors in the epithelium. Bmp4 expression is restricted to villi portions and the inhibition of Bmp4 signaling leads to the formation of crypt along the length of the villi. Therefore BMP4 heterotypic signaling is involved in cell segregation.
In conclusion, subepithelial fibroblasts form together with collagen fibers a network that acts as a structural support of the intestinal morphology. They are contractile cells and their global tension depends on both mechanical and chemical modification of the environment. They are thus able to modify temporarily and locally the mechanical properties of the villi and to adjust nutrients absorption in response to external stimuli. In addition, subepithelial fibroblasts might induce epithelial cell migration as they co-migrate with epithelial cells. They are also involved in the maintenance of proliferative and the spatial segregation between differentiated cells and proliferative cells through paracrine signaling. Finally, they also have an indirect action on intestine homeostasis due to the ECM components they secrete.

The role of the epithelial environment on intestinal homeostasis

In the previous sections we have described the importance of dynamic cell-cell interactions, both homotypic and heterotypic, in the maintenance of stem cells, cell differentiation and migration homeostasis. However epithelial cells also interact with ECM and, although it seems inert, its chemical composition and mechanical properties play a major role in intestinal homeostasis.

Basement membrane

In the histological description of the intestine, we have reported that the epithelium relies on the basement membrane at its basal pole. In addition to the two major component (type IV collagen and laminine) introduced previously, entactin/ nidogen, heparin sulfate proteoglycan, fibronectin, tenascin and type III collagen also constitute the basement membrane (Louvard, Kedinger, and Hauri 1992; Trier et al. 1990). Simon-Assman et al (P Simon-Assmann et al. 1988) proved that basement membrane constituents originate from both mesenchymal and epithelial cells by hybridizing a mouse epithelium with a chicken endoderme. The hybrid was left to grow in nude mice and the immunolabelling revealed that the matrix formed at the frontier between the two cell types was of both origins. This global observation can be refined depending on basement membrane constituents as it was established that collagen IV had a mesenchymal origin whereas heparin sulfate was exclusively synthesized by epithelial cells (Patricia Simon-Assmann et al. 1990). The case of laminin is more complex since the chains constitutive of laminin molecule are of various origins: laminin α1 chain is expressed by the epithelium and α2 by the mesenchyme (Teller et al. 2007).
At the adult stage the constitution of the basement membrane is stable in time compared to the massive changes experienced during the embryonic development (§ embryonic morphogenesis/ ECM composition). Although its global composition is stable the constituents of the BM are constantly renewed. Its renewing was first supposed to precede the migrating epithelial cells BM so that it acts as a treadmill that conveys epithelial cells from the bottom of the crypt to the top of the villi. The study of Trier et al (Trier et al. 1990) disproved this hypothesis: they injected an antibody against laminin in mice and observed that laminin turnover happens in period of weeks whereas epithelial cells are renewed in few days. They also noticed that laminin renewing was not homogeneous in the BM but occured more in a mosaic of new laminin patches among old ones.
The renewing of the basement membrane has to be perfectly orchestrated between epithelial and mesenchymal cells since the composition of the basement membrane is not homogeneous along the crypt villus axis. Indeed as shown in Figure 15 the distribution of basement membrane constituent is topographically defined. As an example laminin α2 is located in the crypt while laminin α3 and α5 are expressed in the villi (Teller et al. 2007). This specific distribution can be correlated to the spatial expression of adhesion receptors in epithelial cells (Figure 15). For instance, integrin subunit (β4A) is present in intestinal epithelium under two distinct forms: one functional and one immature that are spatially distributed so that the pattern of functional subunit coincides with the repartition of its ligand, laminin α5, which is restricted to the villi (Basora et al. 1999). This spatial heterogeneity in the composition of basement membrane is transduced in the cell cytoplasm through transmembrane receptor. The basement membrane composition is thus supposed to play a significant role in the maintenance of stem cell, the induction of migration or differentiation, but it seems difficult to be experimentally proven. Benoit et al (Benoit et al. 2012) succeeded in showing that the loss of RGD interaction (composed of extracellular fibronectin, transmembrane integrin and intracellular vinculin) increased cell migration out of the crypt and affects cell survival signaling in the crypt. RGD signaling would thus be needed to preserve the integrity of the crypt. The differential composition of basement membrane might also drive the apoptosis at the top of the villi. Indeed, fibronectin provides great cell adhesion and is expressed as a decreasing gradient from the bottom of the crypt to the top of the villi while tenascin, to which cells adhere less, exhibits the opposite pattern (Beaulieu 1992; P. Simon-Assmann et al. 1995). These complementary distributions might induce the cell shedding process resulting in the complete loss of cell-matrix adhesion at the villi top. Matrix detachment induces a rapid loss of cell-cell adhesion mediated by E-cadherin and is responsible for anoikis (Beaulieu 1992).
Figure 15: Schematic representation of the heterogeneities of distribution of adhesion molecules in the BM and cell adhesion receptors along the crypt-villus axis. Imported from(Benoit et al. 2012).
To conclude, basement membrane is secreted by subepithelial fibroblasts together with epithelial cells and presents a complex spatial composition. This spatial heterogeneity in its chemical composition might play a key role in tissue homeostasis. However, regarding intestine mechanical properties, it has been demonstrated that the contribution of basement membrane to the mechanical properties of the intestine is negligible(V. I. Egorov et al. 2002).

Mechanical properties of intestine wall

The mechanical strength of the intestine is of major importance; since it ensures the maintenance of the topography (villus & crypt) of the epithelium. Egorov et al (V. I. Egorov et al. 2002) estimated the tensile strength of each layers starting from cadaveric gut that had their intestinal wall sectioned. They sutured back the layers (lamina propria, submucosa and muscular layers) independently of each other to evaluate the strength of each layer and compared to the sutured entire wall. They concluded that the submucosa contributes for up to 70 to 75% to the mechanical strength of the intestine structure while muscular layer contributes only for 15-20%. The input of lamina propria is quite negligible and counts for 5-10% (V. I. Egorov et al. 2002). The mechanical properties of the submucosa are related to the collagen lattice. In the lattice, collagen fibers are arranged into two arrays of collagen fibers interweaved to form a unified sheet (Figure 16). This organization confers more strength than aligned collagen fibers. It also provides some elasticity, as it can undergo peristaltic motion, even though it is composed of non-elastic collagen fibers (Terumasa Komuro 1988).
Figure 16: Arrangement of collagen fibers in the lattice of the submucosa. Scanning electron microscopy micrographs of: a) Global structure of the lattice with the traces of blood vessels (*) (× 80) and b) 300 fold magnification of the two interweaved arrays of collagen fibers. Imported from (Terumasa Komuro 1988).
Collagen organization varies as a function of the intestine localization. In the lamina propria, collagen fibers together with fibroblasts form a loosely arranged network in the villi with a moderate amount of collagen. On the opposite, in the crypt region, pericryptal fibroblasts are arranged into two to three layers, collagen fibers are thicker, more numerous and arranged circumferentially around the crypt (Parker, Barnes, and Kaye 1974). The mechanical tension generated by the contracted myofibroblasts at the crypt base permits the expulsion of cryptal luminal content in the intestinal lumen. In the villi fibroblasts together with the smooth muscle cells oriented longitudinally can contract and retract the villi. Meanwhile the interstitial pressure generated by the blood flow in the capillaries of the villi tends to extend the villi. These two opposite forces generate a dynamic inward tension that avoids the collapsing of the villi. In addition the extension of the villi related to blood pressure might facilitate the upward migration of epithelial cells (Hosoyamada and Sakai 2005).
In the intestinal wall, the mechanical properties of the different layers are combined to ensure the peristaltic motion, to hold and maintain the 3D structure of the epithelium. However the exact influence of the topography of the lamina propria on the epithelium dynamic has never be demonstrated experimentally.
In this first part of the introduction, the dynamic driving the epithelium renewal has been described.

Table of contents :

CHAPTER 1: INTRODUCTION 
I Introduction to the intestine 
I.1.Embryonic morphogenesis of the intestine
I.1.1) Heterotypic cell signaling
I.1.2) Homotypic cell signaling
I.1.3) ECM composition
I.1.4) Mechanical forces
I.2 The maintenance of epithelium homeostasis in the adult small intestine
I 2 1) Stem cells and epithelium renewal
I.2.2) Subepithelial fibroblasts and epithelium homeostasis
I. 2. 3) The role of the epithelial environment on intestinal homeostasis
II Introduction to 3D scaffolds for tissue engineering 
II.1 Artificial extracellular matrix
II.1.1) Chemical composition
II.1.2) Hydrogel physical properties
II.1.3) Importance of dynamic hydrogels
II.2 Microstructured 3D environments
II.2.1) Spatially constrained 3D cultures
II.2.2) 3D microstructured hydrogels: towards micro-organs
III In vitro models of intestinal tissues, state of the art.
III.1. Growing intestinal organoids in artificial extracellular matrix
III.2 Gut- on-chip: growing intestinal tissue in microfabricated systems
CHAPTER 2: RESULTS 
I How to engineer a scaffold that meets the specifications fixed by in vivo microenvironment? 
I. 1 Characterization of collagen I matrix
I 2 Structuring the collagen
I 3 Remodeling of the matrix by epithelial cells and fibroblasts
I. 4 How to strengthen collagen 3D structures?
I. 4.1) Semi-interpenetrating polymer networks
α) Hyaluronic acid/ collagen semi interpenetrating network
β) Fibrin/ collagen semi interpenetrating network
I. 4.2 Chemical cross-linking of collagen fibrils
α) Glycation
β) Glutaraldehyde cross-linking
γ) Genipin cross-linking
II How Caco2 cells behave on a microstructured scaffold 
II. 1. Influence of the 3D structure on the spatial location of proliferative cells
II. 2. Location of proliferative cells during the colonization of 3D structures
II. 3Matrix stiffness induced synchronized collective cell colonization of scaffolds
III From in vivo isolated intestinal crypts to an in vitro intestinal epithelium 
III.1. Growing primary intestinal epithelium on microstructured collagen scaffold
III.1.1) Coating strategies as basement membrane substitute
III.1.2) Seeding isolated primary cells on collagen structure
III.1.3) Seeding organoids on collagen scaffolds
III.2. Proliferation patterns of primary cells on collagen scaffolds
III.3. Influence of fibroblasts on the primary epithelial cells growth
CHAPTER 3: DISCUSSION 
I To which extent can one mimic in vivo environment?
II How the mechanical and physical cues of the matrix affect epithelium behavior
II.1 Evaluation of epithelial tissue forces on the structure
II.2 Emergence of collective coordinated colonization induced by the combination of matrix stiffness and topography
II.3 Local rigidity and geometry sensing integrated at the tissue scale regulates spatial positioning of proliferative cells.
II.4. How is our model useful compared to organoids?
CHAPTER 4: CONCLUSION
CHAPTER 5: MATERIAL AND METHODS

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