Building Blocks for Engineering the Small Intestine

Chapter 37

Building Blocks for Engineering the Small Intestine Nicholas R. Smitha, Eric C. Andersonb, Paige S. Daviesa, and Melissa H. Wonga,c a

Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, OR, bDivision of Hematology and Medical

Oncology, Oregon Health & Science University, Portland, OR, cKnight Cancer Institute, Oregon Health & Science University, Portland, OR

Chapter Outline 37.1 Recent Advances in Intestinal Tissue Engineering 37.2 Current Understanding of ISC Biology 37.2.1 Lgr5 37.2.2 Bmi1 37.2.3 mTert

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Bowel transplantation represents the current standard of care for patients suffering from severe intestinal disorders such as short bowel syndrome (SBS). Despite improvements in surgical procedures, there remains an approximate 50% 5-year survival rate for intestinal transplant patients [1]. Currently, the primary barriers to successful organ transplantation are the lack of transplantable tissue, the potential for transplant rejection, and the requirement of lifelong immunosuppression [2]. These challenges highlight the need to fully explore the developing field of intestinal epithelial stem cell biology and their ability for ex vivo expansion of tissue in order to reach the ultimate goal of engineering personalized transplantable intestine from patient-derived cells.

37.1 RECENT ADVANCES IN INTESTINAL TISSUE ENGINEERING Bioengineering of functional tissues represents the next generation of medical technology. Tissue engineering and regenerative medicine harness the power of developmental and stem cell biology to reconstitute new organs that retain all aspects of normal tissue structure, physiology, and function [3]. Recently, great strides have been made in tissue engineering in many organ systems. For the bladder and liver, functional tissues were successfully created

Regenerative Medicine Applications in Organ Transplantation. © 2014 Elsevier Inc. All rights reserved.

37.2.4 Additional ISC Markers 37.3 Advances in In Vitro Expansion of Intestinal Epithelium 37.4 Concluding Remarks References

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by seeding a mixture of progenitor/stem cells on an acellular scaffold, which were subsequently reconstituted ex vivo[4,5]. Natural scaffolds used to engineer these organs have the advantage of their immunocompatibility and capacity to provide important spatial cues to the seeded cells [6]. Similar strategies have been used to bioengineer even more complex tissues, such as the heart [2]. Clinically, autologous bioengineered intestine transplant is the paramount treatment for patients suffering from severe cases of SBS [7]. SBS is a highly morbid condition that affects both adults and children, and is characterized by the failure to adequately absorb nutrients due to lack of intestinal surface area, as a result of previous resection of diseased tissue [8]. In adults, SBS is typically caused by removal of a large portion of intestine due to Crohn’s disease, ischemia, trauma, or tumors. SBS cases in children are linked to developmental defects including intestinal atresia, volvulus, and necrotizing enterocolitis, a condition most common in premature births [9]. In 1997, the estimated number of patients suffering from SBS was 3 4 per million, yet its incidence appears to be on the rise [10]. In 2004 it was estimated that 24.5 in 100,000 live births had SBS [11]. The current treatment option for SBS, total peritoneal nutrition, is insufficient as many patients fail to thrive and succumb to secondary multi-organ failure [12]. Increasing the gastrointestinal absorptive area through intestinal transplant is the only potentially curative

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treatment option for severe SBS cases, but how such a complex tissue can be bioengineered for successful transplant remains a scientific and clinical challenge. The intestine is a complex organ essential for nutrient absorption and as a protective barrier from external insults. To perform these functions, the intestinal tube is composed of multiple cellular layers including the muscularis, a layer of smooth muscle cells that maintains tissue integrity and performs peristalsis, the mesenchymal stromal cell layer, and the functional layer of columnar epithelium. The mucosal layer is exposed to the external environment, therefore to protect against accumulation of DNA mutations, the epithelial layer is continuously replenished every 7 10 days [13]. Further, to increase absorptive surface area, the intestinal architecture features finger-like villus protrusions. The massive epithelial proliferation is fueled within the crypt invaginations by a population of stem cells within a niche at the base of these crypts [14]. This stem cell niche provides a microenvironment that regulates stem cell homeostasis through complex signaling and functional interactions with surrounding mesenchymal and stromal cells [15]. In order to achieve a physiologically functioning bioengineered intestine, not only must all of the normal cell types be present, but the tissue architecture and signaling mechanisms within the niche must also be successfully recapitulated. What are the cellular and biologic components required to successfully generate viable intestine? Current strategies to bioengineer intestine are built on seminal research by the Vacanti group at Massachusetts General Hospital from the 1990s [16]. This group used porous synthetic scaffolds composed of the biodegradable extracellular matrix (ECM) molecule polyglycolic acid seeded with clusters of dissociated rat gut cells termed “organoid units” (see Table 37.1). The organoid units contained all epithelial and mesenchymal cell types normally present in the gut, presumably containing appropriate stem cell populations to regenerate both epithelial and mesenchymal compartments [17]. The seeded scaffolds were then implanted within vascular regions of host animals to regenerate the cellular components of the intestine

TABLE 37.1 Epithelial Cell Preparations for Use in Tissue Engineered Intestine Cell Preparation

Cellular Composition

Small Bowel

and to become vascularized. Here they formed small encapsulated cysts organized with villus and crypt-like regions [16]. Importantly, when these bioengineered intestinal cysts were anastomosed to the intestine of rats subjected to massive bowel resection—a model of SBS—they incorporated by creating a pouch-like structure and resulted in improved weight gain in the animals [18]. While this exciting result supports the application of engineered intestine for transplantation, it represents only the initial step in this promising process. Importantly, this approach has also shown potential application in engineered large intestine, stomach, and esophagus [19 21], as well as multiple organisms including rat, mouse, pig, and dog [17,22,23]. Despite these promising results, significant hurdles impeding progression into the clinical realm remain. Tissue integrity must be maintained in the engineered intestine in order to prove useful for transplantation. The cysts formed from seeded synthetic scaffolds, while histologically resembling mature intestine, failed to retain the tubular architecture of native intestine that will be required for extensive bowel replacement. Due to this caveat, optimization of scaffolding biomaterials has been a major research focus, including the use of natural tissue scaffolds [24]. To prepare an acellular scaffold, intestinal submucosa is decellularized resulting in the remnant ECM. This natural scaffold material retains crypt invaginations and villus protrusions, providing important spatial information for homing of seeded cells [6]. Further studies have sought to improve the efficiency of organoid unit seeding on scaffolds by testing various synthetic scaffold materials, pore sizes, mechanical properties, and inclusion of tethered growth factors [24 26]. An additional hurdle to the gut bioengineering field is the limited ability to expand intestinal epithelial cells in vitro. To date, only disseminated intestine formed into organoid units have been used to successfully seed scaffolds [24]. While use of these organoid units are ideal because of the minimal cellular manipulation, their use is limited by the amount of starting material required to seed sufficient lengths of scaffold. Recent and exciting advances in the ability to expand intestinal stem cells (ISCs) in culture [27 30] as well as the ability to isolate and grow patient-derived ISCs or patient-derived induced pluripotent stem cells (iPSCs) [31] represent a major and exciting advance for the field.

Expandable in vitro?

Organoid Unit Epithelial and Mesenchymal cells

No

Enteroid

Epithelial cells only

Yes

Organoid

Epithelial and Mesenchymal cells

Yes

37.2 CURRENT UNDERSTANDING OF ISC BIOLOGY The recent identification of novel ISC markers [32 40] and the ability to generate intestinal epithelium from iPSCs [31], coupled with a robust in vitro culturing system [28,30], has revitalized the ISC field. Continued

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Villus

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Differentiated cells

TA cells

+4

Stem cell (mTert+, Bmi1+, Hopx+, Dcamkl1+)

Crypt

Stem cell zone

Paneth cell LRC

CBC stem cell (Lgr5+, Dcamkl1+)

FIGURE 37.1 Architecture of the small intestine and cellular organization of the crypt. The tube of the small intestine is made up of epithelium that is compartmentalized into villus protrusions and crypt invaginations. Stem cells within the stem cell niche at the crypt base give rise to the rapidly proliferating transit-amplifying (TA) cells that differentiate as they migrate up the villus. Intestinal homeostasis is mediated by distinct stem cell populations located in the stem cell zone at the crypt base. The 14 stem cell population represents the slowly cycling, label-retaining cells (LRCs). Markers expressed by cells in this region include: mTert, Bmi1, Dcamkl1, and Hopx. The rapidly cycling crypt-based columnar stem cells (CBCs) are located in the crypt base between differentiated Paneth cells (pink). Markers of CBCs include Lgr5 and Dcamkl1.

development of these approaches now position the bioengineering field to harness these emerging technologies to achieve the ultimate goal of creating autologous patientderived transplantable intestine. This section will highlight these exciting advances in the ISC field and their potential application to bioengineered intestine. The adult intestinal tract displays diversity in structure and function along the cephalo-caudal axis. The small intestine is characterized by finger-like villus projections, lined with differentiated epithelial cell lineages, and adjacent crypt invaginations (crypts of Lieberku¨hn), that house the epithelial stem and progenitor cell populations [14]. In contrast, the adult colon is composed of epithelial-lined crypts, but lacks villi. The differentiated intestinal epithelium in both the small intestine and colon is a single layer of columnar cells composed of four primary cell lineages including absorptive enterocytes, hormone-secreting enteroendocrine cells, mucinsecreting goblet cells, and antimicrobial Paneth cells (the colon lacks Paneth cells) [14]. Under normal homeostatic conditions, the intestinal epithelial cells are regularly and rapidly turned over, with most cell types having a lifespan of approximately 7 10 days in humans—Paneth cells are longer lived, persisting for 2 3 weeks under some conditions [41]. This continual renewal requires a population of multipotent ISCs (or progenitor cells) to self-renew and give rise to the rapidly proliferating population of transit-amplifying (TA) cells (Figure 37.1), precursors to terminally differentiated intestinal epithelial lineages [14].

As a result of the early work of Cheng and Leblond and others [42 44], ISCs have long been known to reside in the base of the crypts. The precise identity and location of these stem cells, however, has proven difficult to elucidate. Early studies by Potten and colleagues postulated that a population of slowly cycling, label-retaining cells (LRCs) existed in the “ 1 4” position relative to the base of the crypt (Figure 37.1) and formed the ISC population [44], while Leblond and colleagues [43] focused on a more rapidly cycling population of cells located in the crypt base which they referred to as the crypt-based columnar cells (CBCs). Despite much work in the field, little progress was made in definitively isolating these cells for confirmation of their stem cell characteristics. However, recent works by a number of laboratories have now discovered cellular and genetic markers expressed in ISCs located at both the 14 position and the crypt base [32 40]. Identification and verification of stem cell activity within these populations have improved our understanding of the location and function of these important cells. Here we review the three primary intestinal epithelial stem/progenitor populations and a number of intriguing minor stem cell populations in the context of proliferative potential for epithelial expansion.

37.2.1 Lgr5 Using lineage-tracing techniques in mouse model systems, Hans Clevers’ group first identified a population

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of cells interspersed among Paneth cells in the crypt base that express the Wnt signaling target gene, leucine-rich-repeat-containing G-protein coupled receptor 5 (Lgr5) [32]. He went on to demonstrate that these Lgr5expressing cells are capable of generating all epithelial lineages of the small intestine and colon [32]. Interestingly, these cells correspond to the original population of CBCs first described by Cheng and Leblond [43]. These Lgr5-expressing cells were found to be a rapidly proliferating population with cycling times on the order of 24 h and thought to undergo symmetric division [13]. This finding was somewhat unexpected as somatic stem cells in other tissues are believed to divide asymmetrically and be relatively quiescent, with long cell cycle times. Further, cells located in the “ 1 4” position were largely negative for Lgr5 expression, suggesting that these two stem cell compartments may differentially contribute to tissue homeostasis [13]. Importantly, the Clevers’ group has also shown that single Lgr5-expressing mouse intestinal cells can be cultured in vitro to expand and generate complete crypt-villus structures [30]. These protocols have now been extended to human cells [29,31,45] as well as to the colon [27,29].

37.2.2 Bmi1 Mario Capecchi’s group employed a similar lineagetracing strategy to characterize the role of the Polycombrepressor complex 1(PRC1) gene Bmi1 in intestinal selfrenewal. Bmi1 is a well known regulator of stem cell behavior in neuronal, hematopoietic, and leukemia cells [36,46 48]. Interestingly, using a tamoxifen-inducible reporter system, they demonstrated that Bmi1 was expressed primarily at the 14 5 position in B10% of crypts [36]. Consistent with the definition of a stem cell population, Bmi1-expressing cells were also able to give rise to all epithelial cell lineages and their presence persisted throughout the life of the mice. Furthermore, ablation of the Bmi1-expressing cells using induced diphtheria toxin-mediated death resulted in rapid epithelial denuding and subsequent death of the animals in 2 3 days, further confirming the essential nature of the Bmi1expressing stem cell population. Building on this work, de Sauvage and colleagues recently demonstrated that ablation of Lgr5-expressing intestinal epithelial cells resulted in the complete loss of Lgr5-expressing cells from the crypt base but did not result in significant histological changes within the crypts [49]. A compensatory increase in Bmi1-expressing ISCs was responsible for epithelial repopulation in the absence of Lgr5-expressing cells. Interestingly, although Lgr5expressing cells were ablated throughout both the small and large intestine, Bmi1-expressing cells contributed to epithelial repopulation only in the duodenum and jejunum

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with little to no expression in the ileum and colon, suggesting that the Bmi1-expressing population is regionally limited. Finally, they also demonstrated that Bmi1-expressing ISCs could give rise to Lgr5-expressing CBCs, supporting the notion of at least two distinct (although potentially overlapping) stem cell populations within the intestine. Under homeostatic conditions—at least in the proximal small intestine—slowly cycling Bmi1-expressing ISCs are upstream of the more rapidly cycling Lgr5-expressing cells. These experiments have important consequences for understanding which populations could respond more rapidly to injury and adequately repopulate the intestinal epithelium.

37.2.3 mTert A third population of ISCs identified by David Breault’s group is characterized by expression of mouse telomerase reverse transcriptase (mTert) [34]. Montgomery et al. found that mTert expression marks a population of slowly cycling LRCs, primarily in the 15 to 18 crypt cell position (consistent with historical reports of the “ 1 4” position). These mTert-expressing cells were identified as single cells in only 1/150 crypts on average, more infrequent than Bmi1-expressing cells, but unlike the Bmi1ISC population, they are found in both the proximal and distal small intestine. While these cells are distinct from the Lgr5-expressing cells, they display some overlap with the Bmi1-ISC population and similarly contribute to all mature intestinal epithelial lineages. Resistance to radiation injury has traditionally been considered a hallmark of stem cells. Paradoxically, cells in the 14 position have long been known to be exquisitely sensitive to low-dose (1 Gy) radiation damage. Lgr5-expressing cells, while resistant to low-dose radiation damage, are sensitive to higher dose (10 Gy) irradiation [32]. However, following both low- and high-dose radiation injury of the intestine, mTert1 cells in the 14 position showed no evidence of apoptosis, suggesting functional heterogeneity of cells in this position. Furthermore, the number of mTert1 crypts increased 12to 15-fold following radiation damage, suggesting that mTert1 ISCs contribute in the regenerative response and restoration of a radiation-sensitive ISC population following injury. Finally, as with the Bmi1-expressing ISC population, mTert ISCs can give rise to Lgr5-expressing cells, again suggesting that they exist upstream of the rapidly cycling Lgr5-ISC population under homeostatic conditions.

37.2.4 Additional ISC Markers Courtney Houchen’s group has characterized an additional discrete population of slowly cycling/quiescent

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Building Blocks for Engineering the Small Intestine

ISCs primarily located in the 14 position which are marked by expression of the doublecortin and CaM kinase-like-1 (Dcamkl1) gene [33]. While these cells often localize to the 14 area, a subset can be found within the Lgr5-expressing population. Unique to the Dcamkl1-expressing ISC population, however, is a spatial expression pattern weighted toward the distal small intestine. Furthermore, these cells are resistant to moderate(6 Gy) but not lethal high-dose (12 Gy) radiation damage [33]. Whether Dcamkl1 cells can give rise to Lgr5expressing cells is currently unknown. CD24, a glycosylphosphatidyl-inositol-anchored membrane protein, which functions in cell cell and cell matrix adhesion and signal transduction has been identified by two different groups as a marker of ISCs [39,50]. This population marks a much broader domain and appears to overlap other ISC populations including those expressing the SRY-box containing gene 9 (Sox9) [51]. Additional markers of putative ISC populations include CD133 [37,40] and Musashi-1 (Msi1) [35]. The precise spatial, temporal, and situational relationship between the ISC populations expressing these various genes remains to be determined. A recent and very intriguing report from Jonathan Epstein’s group has identified a novel marker of ISCs located in the 14 position, the atypical homeobox gene Hopx[38]. Hopx is expressed in 85 90% of intestinal crypts under both homoestatic and injury conditions. However, unlike other 14 position ISC populations, it is expressed throughout the entire length of the small intestine. As expected for a stem cell marker, Hopx1 cells are able to produce all differentiated intestinal epithelial cell types and form organoids under in vitro culture conditions. Interestingly, although under homeostatic conditions Lgr51 and Hopx1 cell populations are distinct, they are able to interconvert— that is, Hopx-expressing cells can give rise to Lgr5expressing cells and vice versa. There is clearly much work to be done to further elucidate the role of Hopx1 ISCs in maintaining intestinal homeostasis and response to injury, but the identification of a bifunctional stem cell population in the intestine provides experimental proof of a mathematical model of ISC function [13] and helps to reconcile divergent behaviors of ISC populations. It appears clear from the available data that multiple subpopulations of stem cells in the small intestine and colon exist and that more remain to be discovered. Under homeostatic conditions, the Lgr5-expressing CBC ISC population appears to be responsible for the majority of epithelial tissue regeneration. Following tissue damage however, a second population of normally slow cycling ISCs characterized by the expression of Bmi1, mTert, Dcamkl1, Hopx, and possibly other genes that have yet to

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be identified, is able to rapidly proliferate in response to injury and repopulate both the homeostatic, Lgr5expressing ISC population as well as the entire intestinal epithelium. Critical issues that remain to be addressed include the spatial, temporal, and hierarchical relationship between the various 14/slow cycling ISC populations and the rapidly cycling Lgr5-expressing CBC population as well as markers of the 14/slow cycling ISC population in the colon. Ongoing experiments in our laboratory and others are expected to answer these questions and provide additional insight into basic ISC biology as well as improving methods for the in vitro generation of viable intestinal epithelium.

37.3 ADVANCES IN IN VITRO EXPANSION OF INTESTINAL EPITHELIUM For nearly 100 years, the prospect of culturing intestinal epithelium to successfully mimic the growth and lineage differentiation that occurs in vivo has been an enigmatic challenge due to the inherent nature of this rapidly renewing cell population and the elusiveness of the ISC. Recently, groundbreaking work from the laboratories of both Calvin Kuo and Hans Clevers reported successful long-term culture of intestinal epithelium [28,30]. While both groups successfully generated in vitro systems to grow and study intestinal epithelium, they took different approaches. Kuo’s group relied upon a mesenchymal niche to drive stem cell maintenance and epithelial differentiation, deriving intestinal “organoids” composed of both epithelial and mesenchymal compartments. Clevers’ group used single epithelial cells conditioned with signaling factors devoid of mesenchymal components to derive “enteroids.” For the purpose of this review, enteroids will refer to in vitro derived intestinal epithelium devoid of mesenchymal components, while organoids will refer to 3D intestinal epithelial cultures that have mesenchymal or stromal components (see Table 37.1). While both of these reports have advanced in the field of intestinal biology immensely, each system has advantages and disadvantages for addressing the ultimate goal of generating intestine for therapeutic repair in patients. The work from Sato and colleagues suggests that programs that dictate intestinal epithelial proliferation and differentiation are cell autonomous [30]. They eloquently demonstrate that only four factors are required to sustain stem cell maintenance, stem cell division, and progenitor differentiation in culture [30]. Their study describes a unique long-term in vitro culture system that can be derived and maintained from mouse isolated small intestinal crypts as well as single intestinal epithelial cells that give rise to intestinal epithelial enteroids [30]. Further, their system suggests that mesenchymal components are

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(A)

(B) Enteroid

Small Bowel

(C) Organoid

Somatic or ES cells Reprogramming

Differentiated cells

iPSCs +Differentiation factors Hindgut endoderm

Lumen

In vitro culture and expansion

Stem cell zone

Organoid

Mesenchyme FIGURE 37.2 Schematic representation of in vitro cultured intestinal epithelia. (A) Enteroids generated by the method of Sato et al. include differentiated villus-like regions (blue) and proliferative crypt-like regions containing a stem cell zone (purple). Apoptotic cells are shed into the inner lumen. (B) Organoids created by [28] contain similar villus and crypt-like domains as the enteroids, but also have an outer mesenchymal layer (pink). (C) The method of [31] generates organoids from reprogrammed human embryonic stem (ES) cells or somatic cells through targeted differentiation into intestinal epithelium, complete with mesenchyme.

not an absolute requirement for the in vitro propagation of intestinal epithelium. However, it has not been determined if epithelia alone could effectively seed and survive on an intestinal scaffold. Most certainly, intestine bioengineered and grown ex vivo would require mesenchymal cell components, however, scaffolds seeded and grown in vivo may be able to utilize native stroma to reseed the mesenchyme to support epithelial growth. The culture conditions described by Sato et al. were designed based on the vast amount of knowledge accumulated over the years regarding important factors necessary to sustain epithelial cells. Knowing the critical role for Wnt signaling in maintaining the proliferative crypt [52 54], the culturing conditions included supplementation with the Wnt agonist, R-spondin-1. In line with the need for effective proliferation, epidermal growth factor was added to the culture milieu [55]. For long-term culture growth and successful passaging, an expansion of crypts was achieved by the addition of Noggin, an inhibitor of bone morphogenetic protein [56]. Finally, because laminin is expressed near the crypt base [57], the authors chose to use an element that has had success in supporting the growth of mammary epithelium, Matrigel, enriched with laminin. Further, since the initial report, additional factors including Wnt3A produced by Paneth cells in the base of the crypt [58] and a Rho inhibitor to inhibit cell death due to anoikis [59] have fine-tuned the culture system. In the cultures initiated from whole crypts, the isolated mouse intestinal crypts underwent fission events to expand into enteroids. Sustained culturing of these isolated crypts resulted in enteroids comprised of numerous crypt domains, encircling a luminal space and interspersed with

differentiated “villus-like” epithelium (Figure 37.2A). Consistent with an in vivo environment, apoptotic cells were shed into the luminal space and Paneth cells were observed to be associated with progenitor cells where new crypt buds formed. Strikingly, the epithelium was organized into a single layer as marked by E-cadherin. Each week, the enteroids could be dissociated and a portion replated, allowing their maintenance in culture for over 8 months. In contrast to the enteroid culture system, Ootani and colleagues approached their culturing conditions in a different manner. They did not specifically isolate the crypts, but rather, they embedded minced tissue from neonatal mouse small or large intestine directly into a 3D collagen gel [28]. Importantly, their preparation included stromal cells, known to be important for establishing a successful crypt niche, and the culture was exposed to an air liquid interface for robust growth. The structures that developed were sphere-like organoids that exhibit proliferation and multilineage differentiation (Figure 37.2B). Similar to the culturing conditions of Sato et al., long-term growth of organoid cultures is also improved by the inclusion of a Wnt agonist. The in vitro organoids can grow for .350 days and are established not only with epithelial cells but also with supporting mesenchymal cells, such as fibroblasts, that aid in the development of a functional gut that can periodically contract, indicating the presence of working muscle cells and neurons. Enteroid cultures produced by Clevers and colleagues lack stromal cells, which were thought to be instrumental in providing a functional ISC niche. Remarkably, their culture conditions promote long-term growth of intestinal epithelium beginning from just a single sorted ISC, although the

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Building Blocks for Engineering the Small Intestine

Autologous cells

557

Seeded scaffold

Bioengineered intestine In vivo regeneration

iPSCs Expand in vitro

+

Natural or

Transplant

+MSCs Crypts

ISCs

Organoid

Synthetic

Ex vivo regeneration

FIGURE 37.3 Potential strategies utilizing stem cells for bioengineering intestine. (Left to right) Autologous patient-derived iPSCs or crypt stem cells are cultured and expanded in vitro to generate sufficient intestinal epithelium in the form of organoids. Single ISC may require the addition of mesenchymal stem cells (MSCs) to regenerate the mesenchymal cell layer of organoids. Organoids are seeded onto synthetic or natural decellularized tissue scaffolds. The seeded scaffolds are then reconstituted in vivo or ex vivo to create viable bioengineered intestine for transplant.

efficiency is quite low (6%). It is interesting that, with the appropriate exogenously supplied signals, ISCs can be cultured and maintained without an explicitly supplied niche. The inclusion of stroma, however, is an advantage of the culture system developed by Kuo and colleagues. This in vitro niche environment allows for the study of the ISC in the context of the niche that exists in vivo. Further, it may also allow for the study of interactions of stem cells with other cell types, such as smooth muscle, endothelium, or neurons, all relevant in vivo interactions, and may prove to be a more viable expansion model for seeding scaffolds for intestinal bioengineering. Finally, the observation that a structural niche is not required for the enteroid culture to thrive in vitro shatters the previous notion that developmental morphogen gradients dictate differential proliferation versus differentiation function among cell populations. It is typically believed that the stem cell must be maintained in a protective structural niche at the crypt base, supplied with subepithelial myofibroblasts and other mesenchymal elements, for structural and signaling support. Sato and colleagues clearly defined culture conditions that allow for a self-renewing intestinal epithelium in the absence of a mesenchymal niche by adding in the necessary mesenchymal components (possibly mesenchymal stem cells capable of regenerating multiple stromal elements or bone marrow derived cells) to allow for adequate growth. Further, the enteroid culture system suggests that the ability to remain a stem cell versus a progenitor or a differentiated cell is a cell autonomous function and not dictated by levels of cell signaling factors. Clearly the ability to grow small organ-like cell clusters may be sufficient for seeding larger scaffolds. However, whether or not the intestinal ECM provides additional important cues for seeding of discrete cells types along the crypt-villus axis during reconstitution of a bioengineered intestine remains to be determined. While a number of different intestinal culture systems bring the promise of expanding a small number of

epithelial cells, the issue of transplant rejection and immunosuppression after transplantation remains if these cells are harvested from nonautologous donors. Recent development in generation of iPSCs and more excitingly, in coaxing these iPSCs toward an intestinal identity bring the promise of autologous bioengineered intestinal transplantation. Using human cells, James Wells’ laboratory recently demonstrated a robust and efficient process to direct differentiation of iPSCs into intestinal tissue [31] (Figure 37.2C). These iPSC-derived organoids were composed of polarized columnar epithelium, surrounded by mesenchyme, which formed villus-like structures and crypt-like regions of proliferative cells and expressed ISC markers. Consistently, all intestinal lineages were represented in this iPSC system. The observation that intestinal mesenchyme differentiation was coordinated with epithelial differentiation indicates that crosstalk between these two regions is important for developing human intestinal organoids. This would agree with the notion that a mesenchymal niche is important for the ISC to reside and thrive in an in vivo setting. The establishment of these culture systems has opened a vast area of research regarding mechanistic questions, exciting real-time imaging, and fine-tuned manipulation that has not been able to occur in the field of intestinal biology until now. The implications these systems have for effectively and successfully expanding intestinal tissue for regenerative therapeutic purposes is eminent. Although the human iPSC system has the advantage over the systems from Ootani and Sato because of the potential to use autologous patient-derived cells, it also has limitations. Components of the in vivo intestine, such as the enteric nervous, vascular, lymphatic, and immune systems are not represented. Thus, there is a need for further improvement to the system, or arguably to define the requirement of other markers and factors essential for establishing and maintaining a functional intestinal epithelium in vitro to provide expansive tissue growth. It does, however, provide

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an exceptional starting point to understand how to regenerate human tissue for therapeutic purposes. [7]

37.4 CONCLUDING REMARKS The recent advances in the ISC field have identified and characterized discrete stem cell populations that are able to be isolated and expanded in vitro. These technologies, coupled with the ability of iPSCs to form de novo (and potentially patient-derived) intestinal epithelium provide a foundation for which these cell types could be isolated from patients for the purpose of engineering autologous intestine for transplant (Figure 37.3). Either patient- or donorderived iPSCs or ISCs would be isolated and cultured in vitro to create organoids. The organoids would be seeded onto either natural or synthetic tissue scaffolds and reconstituted in vivo or ex vivo for subsequent transplant. One important question remaining is what specific cell types are required to successfully seed a scaffold? Given the importance of mesenchymal and stromal cells in stem niche maintenance, these populations will undoubtedly be critical components. A major concern for using in vitro cultured cells is the potential for transformation, and thus a demonstration of the safety of such engineered tissues is essential. Further, future studies will likely center on optimization of the scaffolding materials and methods of in vivo or ex vivo manipulation of the seeded scaffolds. Demonstration of normal physiology and functionality of bioengineered intestine using newly developed assays will surely be a main focus in the near future and help move this technology toward the clinical realm. The effort of multiple groups employing a number of techniques to attain this goal brings excitement to the field and increases the likelihood of success.

[8]

[9]

[10]

[11]

[12]

[13] [14] [15] [16]

[17]

[18]

[19]

REFERENCES [1] Ueno T, Wada M, Hoshino K, Yonekawa Y, Fukuzawa M. Current status of intestinal transplantation in Japan. Transplant Proc 2011;43:2405 7. [2] Orlando G, Baptista P, Birchall M, De Coppi P, Farney A, Guimaraes-Souza NK, et al. Regenerative medicine as applied to solid organ transplantation: current status and future challenges. Transpl Int 2011;24:223 32. [3] Howell JC, Wells JM. Generating intestinal tissue from stem cells: potential for research and therapy. Regen Med 2011;6:743 55. [4] Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet 2006;367:1241 6. [5] Baptista PM, Siddiqui MM, Lozier G, Rodriguez SR, Atala A, Soker S. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology 2011;53:604 17. [6] Baptista PM, Orlando G, Mirmalek-Sani SH, Siddiqui M, Atala A, Soker S. Whole organ decellularization—a tool for bioscaffold

[20]

[21]

[22]

[23]

[24]

Small Bowel

fabrication and organ bioengineering. Conf Proc IEEE Eng Med Biol Soc 2009;2009:6526 9. Levin DE, Dreyfuss JM, Grikscheit TC. Tissue-engineered small intestine. Expert Rev Med Devices 2011;8:673 5. Thompson JS, Weseman R, Rochling FA, Mercer DF. Current management of the short bowel syndrome. Surg Clin North Am 2011;91:493 510. Garg M, Jones RM, Vaughan RB, Testro AG. Intestinal transplantation: current status and future directions. J Gastroenterol Hepatol 2011;26:1221 8. Bakker H, Bozzetti F, Staun M, Leon-Sanz M, Hebuterne X, Pertkiewicz M, et al. Home parenteral nutrition in adults: a European Multicentre Survey in 1997. ESPEN-Home artificial nutrition working group. Clin Nutr 1999;18:135 40. Wales PW, de Silva N, Kim J, Lecce L, To T, Moore A. Neonatal short bowel syndrome: population-based estimates of incidence and mortality rates. J Pediatr Surg 2004;39:690 5. Peyret B, Collardeau S, Touzet S, Loras-Duclaux I, Yantren H, Michalski MC, et al. Prevalence of liver complications in children receiving long-term parenteral nutrition. Eur J Clin Nutr 2011;65:743 9. Li L, Clevers H. Coexistence of quiescent and active adult stem cells in mammals. Science 2010;327:542 5. Potten CS, Booth C, Pritchard DM. The intestinal epithelial stem cell: the mucosal governor. Int J Exp Pathol 1997;78:219 43. Rizvi AZ, Wong MH. Epithelial stem cells and their niche: there’s no place like home. Stem Cell 2005;23:150 65. Choi RS, Vacanti JP. Preliminary studies of tissue-engineered intestine using isolated epithelial organoid units on tubular synthetic biodegradable scaffolds. Transplant Proc 1997;29:848 51. Sala FG, Matthews JA, Speer AL, Torashima Y, Barthel ER, Grikscheit TC. A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse. Tissue Eng Part A 2011;17:1841 50. Grikscheit TC, Siddique A, Ochoa ER, Srinivasan A, Alsberg E, Hodin RA, et al. Tissue-engineered small intestine improves recovery after massive small bowel resection. Ann Surg 2004;240:748 54. Grikscheit T, Ochoa ER, Srinivasan A, Gaissert H, Vacanti JP. Tissue-engineered esophagus: experimental substitution by onlay patch or interposition. J Thorac Cardiovasc Surg 2003;126:537 44. Grikscheit TC, Ochoa ER, Ramsanahie A, Alsberg E, Mooney D, Whang EE, et al. Tissue-engineered large intestine resembles native colon with appropriate in vitro physiology and architecture. Ann Surg 2003;238:35 41. Speer AL, Sala FG, Matthews JA, Grikscheit TC. Murine tissueengineered stomach demonstrates epithelial differentiation. J Surg Res 2011;171:6 14. Agopian VG, Chen DC, Avansino JR, Stelzner M. Intestinal stem cell organoid transplantation generates neomucosa in dogs. J Gastrointest Surg 2009;13:971 82. Sala FG, Kunisaki SM, Ochoa ER, Vacanti J, Grikscheit TC. Tissue-engineered small intestine and stomach form from autologous tissue in a preclinical large animal model. J Surg Res 2009;156:205 12. Gupta A, Dixit A, Sales KM, Winslet MC, Seifalian AM. Tissue engineering of small intestine—current status. Biomacromolecules 2006;7:2701 9.

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Building Blocks for Engineering the Small Intestine

[25] Chen DC, Avansino JR, Agopian VG, Hoagland VD, Woolman JD, Pan S, et al. Comparison of polyester scaffolds for bioengineered intestinal mucosa. Cell Tissues Org 2006;184:154 65. [26] Wulkersdorfer B, Kao KK, Agopian VG, Dunn JC, Wu BM, Stelzner M. Growth factors adsorbed on polyglycolic acid mesh augment growth of bioengineered intestinal neomucosa. J Surg Res 2011;169:169 78. [27] Jung P, Sato T, Merlos-Suarez A, Barriga FM, Iglesias M, Rossell D, et al. Isolation and in vitro expansion of human colonic stem cells. Nat Med 2011;17:1225 7. [28] Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, et al. Sustained in vitro intestinal epithelial culture within a Wntdependent stem cell niche. Nat Med 2009;15:701 6. [29] Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 2011;141:1762 72. [30] Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459:262 5. [31] Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 2011;470:105 9. [32] Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007;449:1003 7. [33] May R, Riehl TE, Hunt C, Sureban SM, Anant S, Houchen CW. Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice. Stem Cell 2008;26:630 7. [34] Montgomery RK, Carlone DL, Richmond CA, Farilla L, Kranendonk ME, Henderson DE, et al. Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells. Proc Natl Acad Sci USA 2011;108:179 84. [35] Potten CS, Booth C, Tudor GL, Booth D, Brady G, Hurley P, et al. Identification of a putative intestinal stem cell and early lineage marker; Musashi-1. Differentiation 2003;71:28 41. [36] Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 2008;40:915 20. [37] Snippert HJ, van Es JH, van den Born M, Begthel H, Stange DE, Barker N, et al. Prominin-1/CD133 marks stem cells and early progenitors in mouse small intestine. Gastroenterology 2009;136:2187 94 e1. [38] Takeda N, Jain R, Leboeuf MR, Wang Q, Lu MM, Epstein JA. Interconversion between intestinal stem cell populations in distinct niches. Science 2011;334:1420 4. [39] von Furstenberg RJ, Gulati AS, Baxi A, Doherty JM, Stappenbeck TS, Gracz AD, et al. Sorting mouse jejunal epithelial cells with CD24 yields a population with characteristics of intestinal stem cells. Am J Physiol Gastrointest Liver Physiol 2011;300: G409 17. [40] Zhu L, Gibson P, Currle DS, Tong Y, Richardson RJ, Bayazitov IT, et al. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 2009;457:603 7. [41] Cheng H. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. IV. Paneth cells. Am J Anat 1974;141:521 35.

559

[42] Cheng H, Leblond CP. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am J Anat 1974;141:461 79. [43] Cheng H, Leblond CP. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am J Anat 1974;141:537 61. [44] Potten CS, Kovacs L, Hamilton E. Continuous labelling studies on mouse skin and intestine. Cell Tissue Kinet 1974;7:271 83. [45] McCracken KW, Howell JC, Wells JM, Spence JR. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat Protoc 2011;6:1920 8. [46] Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 2003;423:255 60. [47] Leung C, Lingbeek M, Shakhova O, Liu J, Tanger E, Saremaslani P, et al. Bmi-1 is essential for cerebellar development and is overexpressed in human medulloblastomas. Nature 2004;428:337 41. [48] Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 2003;425:962 7. [49] Tian H, Biehs B, Warming S, Leong KG, Rangell L, Klein OD, et al. A reserve stem cell population in small intestine renders Lgr5positive cells dispensable. Nature 2011;478:255 9. [50] Gracz AD, Ramalingam S, Magness ST. Sox9 expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro. Am J Physiol Gastrointest Liver Physiol 2010;298. [51] Formeister EJ, Sionas AL, Lorance DK, Barkley CL, Lee GH, Magness ST. Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium. Am J Physiol Gastrointest Liver Physiol 2009;296:G1108 18. [52] Korinek V, Barker N, Willert K, Molenaar M, Roose J, Wagenaar G, et al. Two members of the Tcf family implicated in Wnt/betacatenin signaling during embryogenesis in the mouse. Mol Cell Biol 1998;18:1248 56. [53] Kuhnert F, Davis CR, Wang HT, Chu P, Lee M, Yuan J, et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc Natl Acad Sci USA 2004;101:266 71. [54] Pinto D, Gregorieff A, Begthel H, Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev 2003;17:1709 13. [55] Dignass AU, Sturm A. Peptide growth factors in the intestine. Eur J Gastroenterol Hepatol 2001;13:763 70. [56] Haramis AP, Begthel H, van den Born M, van Es J, Jonkheer S, Offerhaus GJ, et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 2004;303:1684 6. [57] Sasaki T, Giltay R, Talts U, Timpl R, Talts JF. Expression and distribution of laminin alpha1 and alpha2 chains in embryonic and adult mouse tissues: an immunochemical approach. Exp Cell Res 2002;275:185 99. [58] Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 2011;469:415 8. [59] Hofmann C, Obermeier F, Artinger M, Hausmann M, Falk W, Schoelmerich J, et al. Cell cell contacts prevent anoikis in primary human colonic epithelial cells. Gastroenterology 2007;132:587 600.