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Regenerative medicine: prospects for the treatment of inflammatory bowel disease This article reviews the current understanding of the processes driving the development and progression of inflammatory bowel disease (IBD), discusses how the dynamic crosstalk between resident microorganisms, host cells and the immune system is required in order to maintain immune homeostasis, and considers innovative strategies that allow the modification or modulation of the intestinal microorganismal community as a potential approach for treating IBD. This article next rationalizes the use of cell-based regenerative medicine as treatment for IBD, discusses the obstacles hindering its success, summarizes some of the results of recent clinical trials employing these therapies, and discusses ongoing work to enhance mesenchymal stem/stromal cells, making them better suited to the task of repairing the damage within the IBD gut. KEYWORDS: cell therapies n complement system n Crohn’s disease n endothelial cells n ephrin receptors n immune evasion n immunomodulation n inflammatory bowel disease n mesenchymal cells n microflora n stromal cells n Toll-like receptors n ulcerative colitis

Graça Almeida-Porada*1, Melisa Soland1, Joana Boura1,2,3 & Christopher D Porada1 Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157‑1083, USA 2 Department of Bioengineering, Instituto Superior Técnico (IST), Lisbon, Portugal 3 IBB-Institute for Biotechnology & Bioengineering, Instituto Superior Técnico (IST), Lisbon, Portugal *Author for correspondence: Tel.: +1 336 713 1630 Fax: +1 336 713 7290 [email protected] 1

Maintenance of the epithelial intestinal barrier integrity is crucial for homeostasis and protection against the multitude of immunogenic, microbial and toxic aggressions that constantly assault the digestive system [1]. Impairment of this barrier’s function has been shown to be one of the critical factors in the pathophysiology of inflammatory bowel disease (IBD) [2]. IBD refers to a group of disorders, such as Crohn’s disease (CD) and ulcerative colitis (UC), which are defined by inflammation of the GI tract and the presence of an uncontrolled immune response. IBD represents a significant and rapidly growing healthcare burden in developed countries, and impacts the quality of life of millions of people. More than 1.5 million individuals in the USA and 2.2 million in Europe suffer from IBD [3,4]. Although IBD can begin at any age, the peak age of onset occurs in adolescents and young adults, requiring, in most cases, a lifetime of suffering, medical care and a predisposition to the development of cancer. Statistics published by the American College of Gastroenterology Task Force show that IBD is responsible for 2.3 million physician visits, 180,000 hospitalizations and costs US $6.3 billion per year [3]. Furthermore, over the long term, a large percentage of patients with IBD will require surgery for IBD-related complications, further adding to the socio economic impact of this disease(s). Currently, the available therapeutic approaches for IBD rely largely on generalized immunosuppression,

and are intended to induce remission and/or to prevent relapse; no treatment thus far has led to cure of these diseases [5–7]. Furthermore, the side effects, toxicity and lack of response in some patients to existing treatments make the identification/development of a permanent cure imperative. In order to heal the deep lesions and penetrating injuries of the IBD gut and restore homeostasis of the intestinal barrier [8], a regenerative medicine approach using cell-based therapies is currently viewed as one of the most promising options on the horizon for the curative treatment of IBD [9]. In the present paper, we review the current understanding of the processes driving the development and progression of IBD, summarize some of the results of recent clinical trials employing cell-based regenerative medicine as treatment for IBD and discuss some of our own ongoing work to enhance mesenchymal stem/stromal cells (MSCs), making them better suited to the task of repairing the damage within the IBD gut.

10.2217/RME.13.52 © 2013 Future Medicine Ltd

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Etiopathophysiology of IBD The etiopathophysiology of IBD is an active area of investigation. While genetic factors make major contributions to the pathogenesis of IBD, both environmental and developmental factors have been shown to impact the outcome [10]. One key aspect of the human GI tract that is currently receiving a great deal of attention, both in normal biology and in the context of IBD, is the complex ISSN 1746-0751

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and diverse microbial ecosystem referred to collectively as the microbiome. The microbiome is now recognized to exert important protective, metabolic and trophic functions [11], yet the true extent of microbial biodiversity within the intestine is still not known, because many of these organisms cannot be successfully cultured ex vivo. Culture-independent molecular methodologies, such as the analyses of the sequence diversity in the variable regions of 16S rRNA, have been successfully exploited [12] to identify >400 species, the most abundant of which are members of the genus Bacteroides, anaerobic Gram-positive cocci, namely Peptostreptococcus sp., Eubacterium sp., Lactobacillus sp. and Clostridium sp. [13]. Another major challenge in the study of the intestine microbiome resides in the wide spatial and temporal variation seen within different regions of the intestine and also between individuals, ages, cultures and sexes [12]. Despite the beneficial effects exerted by the gut microbiome, a strictly regulated dynamic crosstalk between resident microorganisms and the host luminal antigens, intestinal epithelial cells and immune system is required in order to maintain immune homeostasis [9]. The intestinal immune machinery must simultaneously defend against invading pathogens and prevent intestinal microbe overgrowth, while maintaining a state of immune tolerance towards resident intestinal microbiota [14]. A dysregulation in this balance, and consequently a breakage of immune tolerance towards intestinal microorganisms (native and/or pathogenic), is thought to induce chronic inflammation, and thereby contribute to the development of IBD, especially in genetically susceptible individuals and/or under specific environmental stimuli (e.g., diet and antibiotics) [11,14,15]. Furthermore, although increasing evidence indicates that the microbiome plays an important role during intestinal inflammation, no specific microorganism has been identified as causing/triggering IBD [16]. Nevertheless, alterations in the microflora, such as a decrease in Firmicutes such as bifidobacteria, Lactobacillus and Faecalibacterium prausnitzii, and an increase in mucosal-adherent bacteria, have been verified in IBD-susceptible hosts [17]. The mechanisms by which the microbes or their components interact with the GI tract is still not very well understood; however, emerging evidence is indicating that Toll-like receptors (TLRs), a family of pattern recognition receptors, play a pivotal role in rapid microbial recognition, allowing efficient maintenance of immune tolerance and homeostatic balance of 632

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the intestinal mucosa [14,18–20]. TLRs recognize specific conserved regions or molecular motifs of microorganisms, referred to as pathogen- associated molecular patterns [14,18], leading to the activation of NF- κB and other immuneresponsive genes [21]. Following TLR activation, inflammatory cytokines and chemokines are secreted, and the adaptive T- and B‑cell responses, necessary for the protection of intestinal barrier integrity and elimination of invading microorganisms, are initiated [14,22]. Furthermore, it is also thought that the strategic distribution of TLRs within the surface epithelia has a significant effect on whether resident microbiota will be recognized and, if so, whether that recognition drives proinflammatory responses or represses them [23]. Within IBD-susceptible hosts, an altered TLR pattern has been implicated in the observed overactive T‑cell-mediated inflammation towards microbial antigens, thus leading to amplification and perpetuation of aggressive immune responses that contribute to tissue damage and consequently chronic inflammation [22]. In this context, TLR modulation could provide a valid interventional target for novel therapy development. Given the importance of the microbiome in IBD pathogenesis, it stands to reason that the development of innovative strategies that allow the modification or modulation of the intestinal microorganismal community could hold promise for the treatment of this disease. Indeed, the use of probiotics (beneficial bacteria) or prebiotics (dietary components that foster the growth of beneficial bacteria) has provided significant beneficial effect in several in vivo models, by altering the microbial content, eliminating toxic compounds, enhancing tolerance or increasing epithelial barrier integrity. Recent studies using genetically engineered bacteria that release antiinflammatory agents locally showed that this approach yielded enhanced efficacy in IBD animal models, when compared with systemic administration of the same anti-inflammatory agent [16,19]. Unfortunately, however, data from well-conducted clinical trials to demonstrate efficacy of bacterial-based treatments in humans with IBD are lacking [17,24,25], and the therapeutic benefit of this strategy appears, at least thus far, to be restricted to remission maintenance of the disease [25]. A wealth of data now exist to support the conclusion that abnormalities of the innate and the adaptive immune system are, without a doubt, central to the inappropriate and largely future science group

Regenerative medicine: prospects for the treatment of inflammatory bowel disease

unregulated immune response observed in the gut microbiome in IBD [26,27]. CD4+ T cells seem to play a fundamental role in both CD and UC; however, in CD, the T-cell response is a Th1- and Th17-mediated process, whereas in UC the T-cell profile is characteristic of a Th2 response [27]. Regardless of the specific T-cell subsets involved however, the end results are the same: a poorly regulated T-helper response, a decrease in T-cell apoptosis in the lamina propria, increased numbers of local dendritic cells (DC) and recruiting of cellular inflammatory infiltrates leading to ulcerations of the mucosa and inappropriate and/or delayed healing. Despite the central role of cells classically viewed as part of the immune system play in the proinflammatory/hyperimmune state present in IBD, studies have also demonstrated alterations that are present within other cells of the gut, which may also be of importance to the etiopathophysiology of IBD. Specifically, when functioning as nonprofessional antigen-presenting cells, it appears that intestinal epithelial cells in the IBD gut are rendered unable to promote the expansion of Tregs and thereby inhibit the immune response [26]. It has also been shown that endothelial progenitor cells (EPCs) exist in decreased numbers in the peripheral blood of these patients. This is somewhat paradoxical, given that the presence of an inflammatory state is normally thought to be conducive to high levels of circulating EPCs [28]. This is a potentially important, as yet unexplored, avenue in the context of IBD, given that intestinal microvascular and endothelial cell dysfunction can lead to persistent tissue hypoperfusion and ischemia, and would thereby be expected to contribute to chronic inflammation [28,29]. Furthermore, altered stromal/mesenchymal cells of the intestinal lamina propria may also play a role in perpetuation of intestinal inflammation and contribute to tissue fibrosis, as they express TLRs and surface markers that are used for regulation by professional immune cells [30]. Therefore, current data support the involvement of a large array of altered T, dendritic, endothelial and mesenchymal cells in IBD pathogenesis, suggesting that a curative therapy will require an approach that not only corrects the immune dysfunction, but also stimulates and provides tissue repair [31].

Regenerative medicine solutions for IBD Based on the preceding discussion, it is perhaps not surprising that cell-based therapies have emerged as promising candidates for the future science group

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treatment of IBD with the potential to induce and sustain the state of remission, regenerate the mucosal epithelium and reconstitute the normal function of the intestine. One of the first cellular- based therapies to be explored for the treatment of IBD was hematopoietic stem cell (HSC) transplantation. Interestingly, HSC transplantation was not initially considered a potential treatment for IBD, likely owing to the significant morbidity and mortality that can be associated with this procedure. Rather, several IBD patients who suffered from concurrent malignant conditions, such as non-Hodgkin’s and Hodgkin’s lymphoma, breast cancer and leukemia, underwent HSC transplantation for their malignancy and subsequently reported IBD remission. Since 1993, more than 25 IBD patients who received HSC transplants have experienced longer periods of time (6 months to >5 years) without clinical symptoms, in absence of any medication for IBD. In addition to longer remission periods, mucosal healing was also observed in these patients. This effective HSC-based therapy consisted of ablating the patient’s endogenous hematopoiesis followed by hematopoietic repopulation with autologous/allogeneic HSCs. HSC repopulation eliminates autoreactive T cells as well as memory T cells present in the patient, thus bringing about an immediate halt of the inflammatory reaction, as well as making possible the regeneration of self-tolerant T cells [32–36]. Although autologous HSC transplantation initially exhibited lower risk/toxicity than allogeneic transplantation, improvements in the technique, such as using an alternate source of HSCs (e.g., mobilized peripheral blood rather than bone marrow [BM]), reduced conditioning regimens and a more accurate selection of patients, have greatly reduced the risks associated with allogeneic HSC transplantation. This is a critical advance, since if the patient carries gene mutations that predispose him/her to IBD development one would obviously want to repopulate the patient with new HSC from a healthy donor with no known IBD-associated gene mutations. If successful, allogeneic HSC transplantation could correct the genetic predisposition to the disease and reset the inflammatory/immune reaction by generating new self-tolerant lymphocytes. Even though the results obtained to date with HSC transplantation are very promising, new approaches to improve the procedure are underway. Currently, a PhaseIII clinical trial for CD is being carried out in Europe to evaluate the clinical benefits of HSC mobilization www.futuremedicine.com

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followed by high doses of chemotherapy and autologous HSC transplantation compared with HSC mobilization and best clinical practice for IBD. There are 45 patients enrolled in this study, and the results should be available at the beginning of next year. More advances have also been achieved in the past 2 years in terms of understanding the immunological changes that IBD patients experience following HSC transplantation. Clerici et al. reported the first study showing that levels of proinflammatory cytokines such as TNF‑a, IFN‑g and IL‑12 were reduced after HSC transplantation when patients responded partially, and were reduced even more significantly when patients had a full response [37]. Researchers also found that intestinal TLR4+CD14+ cells diminished in number, suggesting a shift towards tolerance to the GI tract microbiome. In addition, this study also demonstrated a substantial increase in the CD4+CD25+FoxP3+ Treg numbers in the full responders compared with partial responders [37]. Collectively, these findings support the conclusion that HSC transplantation can modulate multiple immunoregulatory pathways. Despite these promising results over the years, it is important to note that HSC transplantation is still a relatively high-risk procedure, given the toxicity of current conditioning regimens, and the risk of graft-versus-host disease (GVHD) in the case of allogeneic transplant, which could be disastrous in the context of the inflammation/ damage already existing within the gut of IBD patients. These shortcomings have fueled the ongoing search for other cell populations that could be used to treat IBD. One such population that has received a great deal of attention over the last 10+ years is the so-called MSC. MSCs have several properties/abilities that have led them to be currently viewed as one of the most promising agents for use in cell therapy for a variety of diseases. Two of the most important of these properties are as following: first, following systemic infusion, MSCs selectively home to areas of inflammation and injury; second, MSCs release soluble factors that modulate immune activity and promote healing by inhibiting apoptosis and stimulating the repair program of resident stem/progenitor cells [38,39]. Another advantage of MSCs over other putative cellular therapeutics is their ability to be harvested safely and relatively noninvasively from either BM or fat. Moreover, they can then be expanded exponentially in vitro without losing their original potential, making it possible to obtain the large numbers of cells necessary 634

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for clinical applications from a relatively small quantity of starting material. Importantly, the safety and efficacy of MSCs in the context of an aberrant immune response has been demonstrated in the clinical arena in patients with GVHD. In this setting, MSCs have been shown to have a broad immunoregulatory role, resulting in significant clinical improvement in multi ple small-scale clinical trials as well as a recent Phase II clinical trial [40–43]. Results from an ongoing placebo-controlled Phase III trial have not been as clear-cut; however, this may perhaps be owing to the high percentage of patients with skin GVHD who already responded well to the current standard of care [44,45]. Looking specifically at IBD, studies in CD, using autologous, adipose-derived MSCs, have proven the beneficial effects of these cells in healing enterocutaneous fistulae after local injection [46,47]. From this Phase I study, six of eight patients (75%) achieved complete closure of the fistula 8 weeks after receiving 3–30 × 106 cells, while the remaining 25% still benefitted, however, the closure was incomplete at this time point [46]. No adverse events were observed during the 1‑year of follow-up, after treatment. Based on these promising results, a more extensive Phase II study involving 49 patients was undertaken [48]. Patients were divided into two groups: the first group received fibrin glue (control: 25 patients) and the remainder received fibrin glue in combination with 20 × 106 adipose MSCs (experimental: 24 patients). From the 24 patients treated with MSCs, 46% experienced complete fistula healing at 8 weeks; the remaining patients in this group were then given a second injection of 40 × 106 adipose MSCs, which enabled an additional 25% to achieve complete healing. By contrast, of the patients receiving fibrin glue alone, only 8% healed after the first treatment, and an additional 8% healed after a second fibrin glue treatment. These results strongly suggest that the healing process is significantly enhanced when adipose MSCs are infused compared with administration of fibrin glue alone. However, presumably owing to the small number of patients, the marked difference was not found to be statistically significant. To further investigate the efficacy of MSCs in this setting, Guadalajara et al. performed a longterm follow-up of these same patients, 4 years after the treatment [49]. Only 21 of the patients originally treated with adipose MSCs and 13 of the patients treated with fibrin glue alone participated in this follow-up study. Twelve of the 21 patients treated with adipose MSC plus future science group


fibrin glue reported a complete closure of the fistula, compared with only three of the 13 in the fibrin glue-alone treatment group. These results again strongly support the addition of adipose MSCs to fibrin glue to enhance healing of CD fistulas. The results for a Phase III clinical trial were released the following year [50]. This study enrolled 200 patients from 19 centers in Spain. Patients were divided in three groups: group A received 20 × 106 adipose-derived MSC alone; group B received 20 × 106 adipose-derived MSCs in addition to fibrin glue; and group C received only fibrin glue. At 12 weeks, 26.56, 38.33 and 15.25% of patients in group A, B and C achieved complete closure of the fistula, respectively. Surprisingly, and in similarity to the Phase II trial, this study, despite clearly highlighting the potential therapeutic value of MSCs, did not show a statistically significant difference among the groups. While results obtained with adipose MSCs have been uniformly promising to date (albeit not statistically significant), studies performed with autologous BM MSCs in CD have thus far provided contradictory results. Ciccocioppo et al. performed local injections of autologous MSCs in ten patients that suffered from fistulizing CD, one with enterocutaneous fistulas and the remainder with perianal fistulas [51]. At 1 year after the transplant, seven patients experienced complete closure of the fistula, while the remaining three achieved a 50% reduction in fistula size. Moreover, seven of the nine patients with perianal fistulas demonstrated healing. Recently, Liang et al. also tested allogeneic BM MSCs as a treatment for IBD in seven patients, injecting the MSCs intravenously at a dose of 1 × 106 cells/kg bodyweight [52]. At 3 months post-treatment, five of the seven patients had achieved remission, which persisted for 2 years in two of these patients. All patients experienced a reduction in diarrhea frequency, as well as in abdominal pain, further highlighting the potential of allogeneic BM MSCs for the treatment of IBD. Although the results are promising, one must interpret them with a degree of trepidation until they are reproduced/validated with a larger sample size, and appropriate controls are included [52]. Similarly, de la Portilla et al. reported the results from a multicenter Phase I/IIa clinical trial using allogeneic adipose-derived MSCs [53]. Twenty four patients received 20 × 106 cells, administered locally in the fistula. At week 12, patients were evaluated and, in the event that the fistula was not completely closed, the future science group

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investigators provided a second dose of 40 × 106 adipose MSCs. From the total patients, 69% showed a reduction in the number of fistulas and 56% a complete fistula closure. None of the patients presented relapse during the first 12 weeks, but five did at some point during weeks 13–24. In addition, two patients experienced serious adverse events that may have been related to the cell infusion procedure. As with the trial carried out by Liang and colleagues [52], this study by de la Portilla et al. needs to be repeated with a larger number of patients and with appropriate controls to confirm the efficacy of the treatment and elucidate the cause of the observed adverse events [53]. In stark contrast to the previously highlighted trials, Duijvestein et al. analyzed intravenous injections of 1–2 × 106/kg bodyweight autologous BM MSC per dose, and administered two doses, spaced 7 days apart [54]. None of the patients experienced remission, and only three patients experienced a decrease in the frequency of soft stool. Possible explanations for why administration of MSCs in the same disease setting can result in such different outcomes are likely the large discrepancy in cell dose between the various studies as well as the route of administration of the MSCs [55]. Cell dosing is an important factor to consider when planning for any cell therapy, as the infused number/dosage often needs to exceed a certain, poorly defined threshold in order to achieve a therapeutic effect.

Increasing homing of MSCs to the intestine One of the touted advantages of using MSCs as therapeutic agents resides in their ability to selectively home to areas of injury and/or inflammation following systemic infusion [36], yet it is not known what percentage of intravenously injected cells actually home to the inflamed area of the intestine/perianal region to exert their therapeutic properties. However, preclinical and some of the clinical studies highlighted above suggest that, in the case of IBD, the natural aptitude of MSCs to migrate in therapeutic numbers to the site of damage may not be adequate. Increasing the number of cells homing to the intestine could thus be a promising option for improving cell therapies for IBD. For example, when using MSCs genetically modified to express the SDF-1 receptor, CXCR-4, in a radiation enteritis model in which SDF-1 was found to be upregulated at the site of injury, much higher levels of intestinal MSC engraftment were obtained [56]. Similarly, Ko et al. showed, in a murine model, that coating www.futuremedicine.com

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MSCs with antibodies against MAdCAM and VCAM-1 prior to infusion improved the homing of these cells to the colon, and thereby enhanced their therapeutic properties [57]. These prior studies thus support the conclusion that the identification of populations of cells that can home preferentially to the intestine and/or have the intrinsic ability to restore the integrity of the intestinal mucosa would greatly increase the probability of success with cell-based therapies for IBD. Ephrins and Eph receptors, both membrane-bound proteins, are differentially expressed in intestinal mucosa, both in humans and mice, with Eph receptors localized to the intestinal crypt region and Ephrin proteins colonized in the villi. The signaling between Ephrin and Eph receptors is restricted to sites of direct cell-to-cell contact and is bidirectional in nature, since it is transmitted in both the receptor-expressing as well as in the ligand-expressing cells [58]. In addition to the role of promoting proliferation of the intestinal epithelium, EphB receptors have been recognized to function as tumor suppressors by controlling cell migration and inhibiting tumoral invasive growth [59]. Studies have also demonstrated that Eph–Ephrin receptor signaling has a decisive role in tissue repair, acceleration of wound closure and maintenance of homeostasis of the intestinal barrier in adults [60,61]. We recently demonstrated, for the first time, that a subpopulation of MSCs within the BM expresses high levels of EphB2, and that upon intraperitoneal transplantation in a fetal sheep model these cells home to the intestine at sevenfold higher levels than their EphB2low counterparts [62,63]. We also demonstrated that the transplanted human cells within the mucosa were positioned mostly within the crypt area, although some could also be found in the villi region. Using antibodies against Musashi-1 and Lgr5, expression of which was absent in MSCs prior to transplant, we showed that the transplanted cells had begun to express specific markers of intestinal lineages. Although the model used to test these cells was a noninjury model, and therefore the inflammatory signals that could induce apoptosis of incoming cells were absent, these studies still showed for the first time that the EphB2high MSC population has an enhanced aptitude for migration to the intestine and, in particular, to the intestinal stem cell region. These results are in agreement with previous reports regarding Eph–Ephrin expression patterns in the intestine, in which it was demonstrated that the proliferative basal crypt cells were EphB2-positive 636

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while the postmitotic differentiated cells were EphrinB1-positive [64]. Based on the key role played by Eph–Ephrin receptor signaling in the repair and maintenance of intestinal homeostasis [60,61], we envision that the use of MSCs selected based on expression of high levels of EphB2 will increase intestinal homing and accelerate the healing of severe intestinal damage, thereby greatly improving the therapeutic efficacy of MSCs in the treatment of IBD.

Correcting the vascular dysfunction in IBD Abnormal or inadequate vasculogenesis, local inflammation and severe epithelial damage are common features of IBD. Since EPCs represent a promising tool for ischemic cardiac and vascular repair, we investigated whether transplanted EPCs can contribute to the intestinal vasculogenic process and/or the stem or mature epithelial cell pools. In order to study the intrinsic ability of human EPCs to contribute to the epithelial or vascular bed of the small intestine, we transplanted preimmune fetal sheep with human umbilical cord blood-derived endothelial colony forming cells (ECFCs), provided by Yoder and colleagues [65]. Prior to transplant, ECFCs expressed CD31, CD105, CD144 and CD146, and were negative for CD45. Low levels of CD34, CD117 and CD133 were also expressed [66]. We then evaluated the recipients at 85 days post-transplant for the contribution of these cells to the intestinal architecture. ECFCs migrated efficiently to the intestine, and engrafted preferentially within the mucosal layer above the muscularis mucosa, in the area of the crypts of Lieberkühn, at levels as high as 35%. ECFCs constitutively express EphB2, providing a possible explanation for the ability of these cells to migrate to the intestine at such high efficiency, and corroborating our prior work with MSCs. We also examined the ability of these cells to contribute to the stromal/vasculature area of the intestine and found that numerous cells localized to areas of high vascularization, suggesting they could be contributing to the microvasculature. Last, we also showed that approximately 28% of the stromal myofibroblast population in the small intestine in transplanted animals were human-derived. Intestinal myofibroblasts exist as a syncytium with fibroblasts and mural cells in the lamina propria of the gut [67], and express growth factors, prostaglandins and extracellular matrix molecules, mediate information flow between the epithelium and the mesenchymal elements of the lamina propria [67], and play a future science group


vital role in intestinal wound and disease repair. The inherent ability of ECFCs to contribute to the regions of the intestine harboring the stem cell pool, microvasculature and regions responsible for providing trophic support to promote healing and restore homeostasis suggests that these cells could also prove to be valuable therapeutics for regenerating the gut, alone or perhaps in conjunction with MSCs.

MSC immunomodulation & immune evasion In addition to MSCs’ intrinsic capacity to home to areas of injury and inflammation, and to then participate in tissue repair [38,39,68–73], MSCs also fulfill another vital function, which is to dampen inflammation/hyperimmunity present within the damaged/diseased tissue. MSCs possess an impressive inherent ability to modulate multiple components of the adaptive immune system both in vitro [74–81] and in vivo [82–84], inhibiting proliferation and maturation of cytotoxic and T helper cells, DCs, NK cells and B cells, as well as suppressing NK-cell-mediated cytotoxicity. MSCs also stimulate the production of Tregs, which can further dampen any ongoing immune response. MSCs’ immunomodulatory effects are mediated via cell–cell contact and the release of a host of soluble factors, including nitric oxide, inducible NOS, TGF-b1, IL‑10, PGE2, HLA‑G, IDO and HGF. MSCs’ ability to dampen the aberrant immune reaction and/ or ongoing inflammation present within the damaged/diseased tissue greatly facilitates the process of repair/recovery, further enhancing the utility of these cells for regenerative medicine [85]. These pronounced immunomodulatory properties of MSCs are also of direct relevance to IBD, given the key role played by the deregulated immune system in this disease. Indeed, MSCs have been shown, preclinically, to extend skin allograft survival in baboons [86], and clinical trials have demonstrated the ability of MSCs to prevent/eliminate GVHD in human patients [40–42]. In addition to modulating immunity, MSC expression of intermediate levels of HLA class I, and their absence of HLA class II and other costimulatory molecules, led investigators to presume that these cells should be able to evade immune recognition and destruction following allogeneic transplantation [87]. However, studies in both mice and swine have now shown that MSCs are not invisible to the recipient’s immune system, and can trigger immune responses that lead to rejection following transplantation [88–92] future science group

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when an HLA class I mismatch exists between the donor and the recipient [90,91,93]. The brain has long been thought to represent a relatively immune-privileged site, yet, even when allogeneic MSCs were injected intracranially, they induced an NK- and cytotoxic T-cell-mediated immune response in the highly translational rhesus macaque model. This response was found to be dependent on both cell dose and the degree of HLA class I mismatch between the donor and recipient [94]. Since intestinal NK cells and an imbalance in T-cell polarization both appear to play a key role in the pathogenesis of IBD [27,95– 100], it is likely that, even among the cells that reached the intestine in the clinical trials to-date, the vast majority probably could not survive the inflammatory/proapoptotic environment present within the IBD intestine, thus limiting engraftment and precluding the differentiation into intestinal cells that has been observed in animal models [101–105]. In many instances, transplant timing or underlying disease can rule out the use of autologous MSCs. Finding ways to overcome donor–recipient HLA class I mismatch is thus imperative to realize the full therapeutic benefit from allogeneic MSC transplantation. If an approach could be developed that suppressed HLA class I expression on MSCs, an ‘off-theshelf’ MSC-based therapeutic could be created. The availability of such a product would lower the costs associated with a personalized MSC therapy, and eliminate the downtime required to expand the patient’s MSCs, making it possible to treat anyone in need almost immediately. The ubiquitously present human cytomegalovirus (HCMV) possesses several means of evading recognition by NK cells and cytotoxic T lymphocytes (CTLs) [106–109]. HCMV is able to prevent CTL lysis of cells it infects by virtue of its unique short producing (US) proteins. Some of the most important among these proteins are US2, US3, US6 and US11, which work through multiple complementary mechanisms to downregulate surface expression of HLA class I on infected cells [110–112]. We, therefore, undertook studies in which we singly expressed each of these US proteins in human BM-derived MSCs, and evaluated which was most effective at downregulating HLA class I expression, presuming that downregulation of HLA class I should protect MSC from CTL attack. We recently reported that US6 and US11 were most effective at reducing HLA class I surface levels on MSCs, and that this reduction in HLA class I expression, as predicted, substantially decreased both human and sheep lymphocyte proliferation www.futuremedicine.com

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when US-expressing MSCs were used as stimulators in standard mixed lymphocyte reactions [113]. However, HLA class I expression is required to engage class I inhibitory receptors on NK cells and thereby prevent NK-cell lysis. For this reason, we also investigated whether ablating HLA class I expression would increase NKcell killing of the US protein-expressing MSCs. Surprisingly, we found that expression of US11 also protected MSCs from NK-cell lysis. For almost two decades, we have used fetal sheep as a model of human stem cell transplantation, and have reported that human-derived MSCs have the ability to engraft and generate significant numbers of functional hepatocytes in this model [72,114]. At the time of transplant, both CTL and NK-like cells are present in circulation, but engraftment of human cells is possible without additional manipulation, owing to the relative immaturity of the nascent fetal immune system [115]. We therefore used this paradigm of hepatocyte formation to determine whether expressing US6 or US11 would allow higher levels of engraftment of xenogeneic human MSCs, and to ascertain whether expression of these proteins would impact upon the ability of the human MSCs to subsequently differentiate in vivo into liver-specific cell types. Our results showed that US6 or US11 expression significantly increased the levels of engraftment of human MSCs within the fetal sheep liver, while maintaining the ability of the MSCs to be reprogrammed in vivo into cells with the phenotype and functionality of hepatocytes [113]. We have thus developed a viable strategy to evade immunological detection/rejection, and thereby maximize MSC engraftment in mismatched recipients. Since the HLA class I mismatch between the donor and the recipient appears to be one of the major factors responsible for the failure of allogeneic-donor MSCs to engraft [90,91,93,116], we propose that MSCs engineered to express the US6 and/or US11 proteins from HCMV could potentially represent an off-the-shelf product that would eliminate the need for donor–recipient HLA class I matching, making it possible to achieve engraftment of curative numbers of MSCs at the site of injury within the IBD intestine, and thereby improve their therapeutic benefit.

Enhancing survival of transplanted MSCs to increase therapeutic efficacy In addition to the cellular barriers to allogeneic engraftment, transplanted MSCs are also 638

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subject to innate defense mechanisms such as the complement system [117], which plays an essential role in the inflammatory process, and serves as a critical bridge between the innate and adaptive arms of the immune response [118–120]. Upon activation, complement proteins function as chemotactic factors and amplifiers of the inflammatory response, promote the destruction of infectious agents, cause cytolysis of damaged cells and tissues, and play an active role in allograft rejection [121,122]. MSCs express functional receptors for anaphylatoxins C3a and C5a, and these receptors contribute to the recruitment of MSCs to sites of injury [123]. In addition to being recruited to sites of complement activation, MSCs can also activate the complement system, which results in production of soluble C3a and C5a, and deposition of complement-activated molecules on their surfaces [124]. MSCs express soluble factor H and the complement regulatory proteins CD46, CD55 and CD59, allowing MSCs to be able to inhibit activation of the complement system to a certain extent [124–126]. Still, in the presence of an activated complement system, these innate mechanisms of protection are insufficient to prevent cellular damage and death [127]. Specifically within the context of IBD, recent studies have shown that complement blockade results in clinical improvement [128,129]. It is thus possible that despite displaying molecules that confer some protection from complement-mediated lysis, MSCs are still able to be damaged by the activated complement proteins after being recruited to wound sites within the gut, leading to premature cell death and limiting their therapeutic benefit. We recently undertook a study to attempt to enhance the complement resistance of MSCs, with the hopes that this would enable these cells to better survive the harsh proapoptotic environment present within the IBD gut [130]. To develop a means of accomplishing this objective, we again turned to HCMV, which has developed means of evading the complement system within infected hosts, both via incorporating the host-encoded complement inhibitor proteins, CD55 and CD59, into its envelope [131] to increase egress from an infected cell, and by inducing upregulation of the host-encoded CD55 and CD46 after infection [132]. As detailed in the previous section, we and others have previously shown that genetically engineering mesenchymal cell populations, as well as other cell populations, to express the HCMV proteins US2, US3, US6 and US11 downmodulates future science group


HLA class I expression [133–135], leading to a decrease in allogeneic CTL activation and NKcell killing [113]. We therefore investigated the ability of the HCMV US proteins US2, US3, US6 and US11 to protect MSCs from complement lysis. We demonstrated that different US proteins upregulated the expression of complement regulatory proteins at different levels, but that overexpression of US2 protein on MSCs enhanced the production of all of the complement regulatory molecules expressed on these cells [130]. Furthermore, using a complement lysis assay, we showed that expression of US2 on MSCs functionally protected these cells from complement lysis. Expression of HCMV US proteins on MSCs, particularly US2, might thus constitute an additional strategy by which, in mismatched recipients, one could increase MSC engraftment and/or persistence in the injured tissue in order to release trophic factors. Perhaps by combining expression of US6 or US11 with US2, one could further enhance the individual beneficial effects, protecting the MSCs from both T/NK-cell killing and complement-mediated lysis.

Conclusion IBD is a group of diseases with a complex etiopathophysiology, involving altered microbiome, aberrant immunity, destruction of the critical barrier provided by the intestinal epithelium and a compromised microvascular system, the end result of which is a lifetime of suffering and medical care. To date, therapies for IBD have relied predominantly on nonspecific immunosuppression in an effort to halt the immunemediated damage to the intestine. Such an approach can, at best, hope to induce remission, rather than actual provide a cure for IBD. The use of cells (MSCs in particular) as therapeutics for IBD showed great promise in preclinical animal models, with transplanted cells both contributing directly to the regeneration of damaged intestinal tissue and promoting endogenous repair pathways. While results in human IBD patients have been encouraging, MSC-based therapies have thus far fallen short of their goal to provide a permanent cure for IBD. This appears to be due to inefficient homing of the transplanted cells to the site of injury, clearance of these cells by the immune system of the allogeneic recipient, the inability of the transplanted MSCs to survive the harsh, proapoptotic environment of the IBD gut and a failure of therapies to date to restore normalcy to the aberrant immune response present in future science group

Review

IBD patients, preventing further destruction and allowing healing to commence. We and others have recently undertaken efforts to enhance the homing of MSC to the intestine, arm MSCs with defenses against toxic molecules present within the IBD gut and render them less visible to the allogeneic immune system. We envision these efforts ultimately overcoming the limitations that have been observed thus far in cell therapies for IBD, enabling the successful use of MSCs as off-the-shelf curative therapeutics for IBD.

Future perspective The development of successful therapies for IBD has been thwarted by the inability to concurrently target the vast array of complex interacting factors triggering IBD. We put forward that failure to restore an appropriate balance to the delicate crosstalk between microorganisms, host cells and the various players of the innate and adaptive immune systems will preclude the success of any therapeutic strategy aimed at the other aspects of IBD. Therefore, in order to develop a treatment that is curative, the therapeutic approach would need to correct the pathologic triggers, altered immune response and abnormalities present within the parenchymal and stromal components of the IBD gut. Cell regenerative therapies have the advantage of being able to concurrently offer replacement of damaged cells, stimulation of endogenous repair mechanisms and control of the altered immune response, thereby leading to re-establishment of normal gut function. Nevertheless, results thus far indicate that cell therapies have not consistently delivered the expected improvement in patients with IBD; therefore, there is an urgent need to optimized current approaches. To accomplish this objective, it will be necessary to develop a cellular product that, intrinsically or by genetic modification, has the ability to reset the abnormal skewing of immunity, thereby inducing a state that promotes tolerance towards both the endogenous microbiome and host antigens. In addition, the identification and overexpression of molecules promoting cellular homing to the damaged tissue would allow a higher concentration of cells at the site of inflammation to attain the desired therapeutic dosage. Given the widespread damage to not just the intestinal epithelium, but also the supportive stroma and the microvascular network, a curative cell-based therapy may need to rely on a combinatorial approach employing multiple cell types. In support of this suggestion, our own www.futuremedicine.com

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work in the sheep model showed that, in absence of injury, both MSCs and EPCs can migrate to the intestine and contribute to the developing cytoarchitecture, but their patterns of engraftment and the cell types to which they give rise differ. Thus, while MSCs and EPCs may individually be able to address certain aspects of IBD pathophysiology, their combined use could produce a more holistic therapeutic response. Also, if replacement of the damaged tissue is the goal, the infused cells will have to survive inflammation in order to integrate and replace the damaged intestinal layers. Once an appropriate immune milieu has been re-established,

efforts can then be undertaken to restore the rich diversity of the intestinal microflora and to regenerate the damaged intestinal tissue. Financial & competing interests disclosure This work was supported by the SENS Foundation. J Boura is an FCT scholar and supported by SFRH/BD/70948/2010. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Background The current therapeutic approaches for inflammatory bowel disease (IBD) rely largely on generalized immunosuppression, and are intended to induce remission and/or to prevent relapse; no treatment thus far has led to the cure of these diseases. The side effects, toxicity and lack of response to existing treatments in some patients make the identification/development of a permanent cure imperative. Etiopathophysiology of IBD The microbiome plays a role in IBD pathogenesis and, as such, modification or modulation of the intestinal microorganismal community could potentially be a therapeutic target. However, well-conducted clinical trials are needed to demonstrate the efficacy of bacterial‑based treatment in humans with IBD. In addition to the disruption of the epithelial intestinal barrier and alteration of the innate and adaptive immune system, dysregulation of the cellular microenvironmental components such as myofibroblasts, endothelial cells and mesenchymal cells leads to persistent tissue hypoperfusion and ischemia, contributing to inflammation and fibrosis. Therefore, curative therapies for IBD will require approaches that will correct immune dysfunction and stimulate tissues repair. Regenerative medicine solutions for IBD Cell therapies have emerged as promising candidates for the treatment of IBD; however, conflicting results have emerged from the current clinical trials using mesenchymal stem cells. It is possible that factors such as insufficient homing of mesenchymal stem cells to the intestine and inability to survive in an inflammatory environment are contributing to the lack of tissue repair observed after infusion. Therefore, enhancing the numbers and survival of the cellular product reaching the intestine could potentially increase therapeutic efficacy.

References

5

Theis VS, Sripadam R, Ramani V, Lal S. Chronic radiation enteritis. Clin. Oncol. (R. Coll. Radiol.) 22(1), 70–83 (2010).

6

Stenson WF, Snapper SB. Challenges in IBD research: assessing progress and rethinking the research agenda. Inflamm. Bowel Dis. 14(5), 687–708 (2008).

Papers of special note have been highlighted as: n of interest 1

Abraham C, Cho JH. Inflammatory bowel disease. N. Engl. J. Med. 361(21), 2066–2078 (2009).

2

Strober W, Fuss I, Mannon P. The fundamental basis of inflammatory bowel disease. J. Clin. Invest. 117(3), 514–521 (2007).

3

4

Talley NJ, Abreu MT, Achkar JP et al. An evidence-based systematic review on medical therapies for inflammatory bowel disease. Am. J. Gastroenterol. 106(Suppl. 1), S2–S25; quiz S26 (2011). Saleh M, Elson CO. Experimental inflammatory bowel disease: insights into the host-microbiota dialog. Immunity 34(3), 293–302 (2011).

640

7

8

9

Ford AC, Sandborn WJ, Khan KJ, Hanauer SB, Talley NJ, Moayyedi P. Efficacy of biological therapies in inflammatory bowel disease: systematic review and meta-analysis. Am. J. Gastroenterol. 106(4), 644–659; quiz 660 (2011).

moving toward a stem cell-based therapy. World J. Gastroenterol. 14(29), 4616–4626 (2008). 10 Cho JH, Brant SR. Recent insights into the

genetics of inflammatory bowel disease. Gastroenterology 140(6), 1704–1712 (2011). 11 Marsal J, Agace WW. Targeting T-cell

migration in inflammatory bowel disease. J. Intern. Med. 272(5), 411–429 (2012). 12 de Vos WM, de Vos EA. Role of the intestinal

microbiome in health and disease: from correlation to causation. Nutr. Rev. 70, S45–S56 (2012).

Okamoto R, Watanabe M. Molecular and clinical basis for the regeneration of human gastrointestinal epithelia. J. Gastroenterol. 39(1), 1–6 (2004).

13 Canny GO, McCormick BA. Bacteria in the

Lanzoni G, Roda G, Belluzzi A, Roda E, Bagnara GP. Inflammatory bowel disease:

14 Abraham C, Medzhitov R. Interactions

Regen. Med. (2013) 8(5)

Intestine, helpful residents or enemies from within? Infect. Immun. 76(8), 3360–3373 (2008). between the host innate immune system and

future science group


microbes in inflammatory bowel disease. Gastroenterology 140(6), 1729–1737 (2011). 15 Damman CJ, Miller SI, Surawicz CM,

16 Bosani MA, Ardizzone S, Porro GB. Biologic

29 Deng X, Szabo S, Chen L et al. New cell

therapy using bone marrow-derived stem cells/endothelial progenitor cells to accelerate neovascularization in healing of experimental ulcerative colitis. Curr. Pharm. Des. 17(16), 1643–1651 (2011). DW. Intestinal myofibroblasts: targets for stem cell therapy. Am. J. Physiol. Gastrointest. Liver Physiol. 300(5), G684–G696 (2011).

17 Anderson J, Edney R, Whelan K. Systematic

18 Maynard CL, Elson CO, Hatton RD, Weaver

stem cell therapy of intestinal disease: are their effects systemic or localized? Curr. Opin. Gastroenterol. 27(2), 119–124 (2011). immunologic tolerance with no or minimal toxic immunosuppression for prevention of immunodeficiency and autoimmune diseases. World J. Surg. 24(7), 797–810 (2000). reconstitution of antibody responses following transplantation of purified allogeneic hematopoietic stem cells. J. Immunol. 186(7), 4191–4199 (2011).

35 Vanikar AV, Modi PR, Patel RD et al.

Hematopoietic stem cell transplantation in autoimmune diseases: the Ahmedabad experience. Transplant. Proc. 39(3), 703–708 (2007).

37 Clerici M, Cassinotti A, Onida F et al.

data: new insight. J. Pathol. 217(2), 318–324 (2009). 39 Le Blanc K. Mesenchymal stromal cells:

tissue repair and immune modulation. Cytotherapy 8(6), 559–561 (2006). 40 Bacigalupo A. Management of acute graft-

versus-host disease. Br. J. Haematol. 137(2), 87–98 (2007). 41 Le Blanc K, Frassoni F, Ball L et al.

Potential role of Th17 cells in the pathogenesis of inflammatory bowel disease. World J. Gastroenterol. 15(46), 5784–5788 (2009).

Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versushost disease: a Phase II study. Lancet 371(9624), 1579–1586 (2008).

28 Garolla A, D’Inca R, Checchin D et al.

Reduced endothelial progenitor cell number and function in inflammatory bowel disease:


D, Pascual I, Peiro C, Rodríguez-Montes JA. A Phase I clinical trial of the treatment of Crohn’s fistula by adipose mesenchymal stem cell transplantation. Dis. Colon Rectum 48(7), 1416–1423 (2005). 47 García-Olmo D, Herreros D, De-La-

Quintana P et al. Adipose-derived stem cells in Crohn’s rectovaginal fistula. Case Rep. Med. 2010, 961758 (2010). 48 García-Olmo D, Herreros D, Pascual I et al.

Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a Phase II clinical trial. Dis. Colon Rectum 52(1), 79–86 (2009). 49 Guadalajara H, Herreros D, De-La-Quintana

P, Trébol J, García-Arranz M, García-Olmo D. Long-term follow-up of patients undergoing adipose-derived adult stem cell administration to treat complex perianal fistulas. Int. J. Colorectal Dis. 27(5), 595–600 (2012).

38 Caplan AI. Why are MSCs therapeutic? New

26 Singh UP, Singh NP, Singh B et al. Stem cells

27 Liu ZJ, Yadav PK, Su JL, Wang JS, Fei K.

46 García-Olmo D, García-Arranz M, Herreros

Immunomodulatory effects of unselected haematopoietic stem cells autotransplantation in refractory Crohn’s disease. Dig. Liver Dis. 43(12), 946–952 (2011).

25 Steidler L. Genetically engineered probiotics.

as potential therapeutic targets for inflammatory bowel disease. Front. Biosci. (Schol. Ed.) 2, 993–1008 (2010).

mesenchymal stem cells in the treatment and prevention of graft-versus-host disease. Adv. Hematol. 2011, 427863 (2011).

potential in the treatment of inflammatory bowel disease. Clin. Exp. Gastroenterol. 3, 1–10 (2010).

24 Steidler L. Microbiological and

Best Pract. Res. Clin. Gastroenterol. 17(5), 861–876 (2003).

45 Lin Y, Hogan WJ. Clinical application of

36 Swenson E, Theise N. Stem cell therapeutics:

23 Rakoff-Nahoum S, Paglino J, Eslami-

immunological strategies for treatment of inflammatory bowel disease. Microbes Infect. 3(13), 1157–1166 (2001).

dilemma – does a negative Phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy 15(1), 2–8 (2013).

severe autoimmune diseases: progress and problems. Haematologica 83(8), 733–743 (1998).

22 Cario E. Therapeutic impact of Toll-like

Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 118(2), 229–241 (2004).

44 Galipeau J. The mesenchymal stromal cells

34 Marmont AM. Stem cell transplantation for

21 Michelsen KS, Arditi M. Toll-like receptors

receptors on inflammatory bowel diseases: a multiple-edged sword. Inflamm. Bowel Dis. 14(3), 411–421 (2007).

human mesenchymal stem cells added to corticosteroid therapy for the treatment of acute graft-versus-host disease. Biol. Blood Marrow Transplant. 15(7), 804–811 (2009).

33 Linderman JA, Shizuru JA. Rapid

20 Szebeni B, Veres G, Dezsofi A et al. Increased

and innate immunity in gut homeostasis and pathology. Curr. Opin. Hematol. 14(1), 48–54 (2007).

43 Kebriaei P, Isola L, Bahceci E et al. Adult

32 Good RA. Progress toward production of

19 Kucharzik T, Maaser C, Lügering A et al.

expression of Toll-like receptor (TLR) 2 and TLR4 in the colonic mucosa of children with inflammatory bowel disease. Clin. Exp. Immunol. 151(1), 34–41 (2008).

Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 81(10), 1390–1397 (2006).

31 Manieri NA, Stappenbeck TS. Mesenchymal

CT. Reciprocal interactions of the intestinal microbiota and immune system. Nature 489(7415), 231–241 (2012). Recent understanding of IBD pathogenesis: implications for future therapies. Inflamm. Bowel Dis. 12(11), 1068–1083 (2006).

42 Ringden O, Uzunel M, Rasmusson I et al.

30 Mifflin RC, Pinchuk IV, Saada JI, Powell

targeting in the treatment of inflammatory bowel diseases. Biologics 3, 77–97 (2009). review: faecal microbiota transplantation in the management of inflammatory bowel disease. Aliment Pharmacol. Ther. 36(6), 503–516 (2012).

disease (GVHD) by infusion of bone marrowderived mesenchymal stem cells (MSCs), highlighting the clinical value of these cells for immunomodulation and treatment of diseases involving overactive immunity.

a possible link to the pathogenesis. Am. J. Gastroenterol. 104(10), 2500–2507 (2009).

Zisman TL. The microbiome and inflammatory bowel disease: is there a therapeutic role for fecal microbiota transplantation? Am. J. Gastroenterol. 107(10), 1452–1459 (2012).

n

First Phase II study to report successful resolution of graft-versus-host

www.futuremedicine.com

Review

n

One of the longest follow-up analyses on patients who received MSCs for the treatment of Crohn’s disease. Clearly shows the potential of these cells as therapeutics in the context of inflammatory bowel disease.

50 Herreros MD, García-Arranz M,

Guadalajara H, De-La-Quintana P, García‑Olmo D; FATT Collaborative Group. Autologous expanded adiposederived stem cells for the treatment of complex cryptoglandular perianal fistulas: a Phase III randomized clinical trial (FATT 1: Fistula Advanced Therapy Trial 1) and long-term evaluation. Dis. Colon Rectum 55(7), 762–772 (2012).

641

Review


51 Ciccocioppo R, Bernardo ME, Sgarella A et al.

62 Colletti E, Zanjani ED, Porada CD, Almeida-

Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn’s disease. Gut 60(6), 788–798 (2011).

Porada MG. Tales from the crypt: mesenchymal stem cells for replenishing the intestinal stem cell pool. Blood 112, Abstract 390 (2008).

52 Liang J, Zhang H, Wang D et al. Allogeneic

mesenchymal stem cell transplantation in seven patients with refractory inflammatory bowel disease. Gut 61(3), 468–469 (2012).

63 Colletti E, El Shabrawy D, Soland M et al.

EphB2 isolates a human marrow stromal cell subpopulation with enhanced ability to contribute to the resident intestinal cellular pool. FASEB J. 27(6), 2111–2121 (2013).

53 de la Portilla F, Alba F, García-Olmo D,

Herrerías JM, González FX, Galindo A. Expanded allogeneic adipose-derived stem cells (eASCs) for the treatment of complex perianal fistula in Crohn’s disease: results from a multicenter Phase I/IIa clinical trial. Int. J. Colorectal Dis. 28(3), 313–323 (2013). 54 Duijvestein M, Vos AC, Roelofs H et al.

Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn’s disease: results of a Phase I study. Gut 59(12), 1662–1669 (2010). 55 Panes J, Benitez-Ribas D, Salas A.

Mesenchymal stem cell therapy of Crohn’s disease: are the far-away hills getting closer? Gut 60(6), 742–744 (2011). 56 Zhang J, Gong JF, Zhang W, Zhu WM, Li

JS. Effects of transplanted bone marrow mesenchymal stem cells on the irradiated intestine of mice. J. Biomed. Sci. 15(5), 585–594 (2008). 57 Ko IK, Kim BG, Awadallah A et al. Targeting

improves MSC treatment of inflammatory bowel disease. Mol. Ther. 18(7), 1365–1372 (2010). n

Demonstrates the importance of efficient migration of infused cells to the site of injury for therapeutic benefit. Given the pervasive presumption that MSCs naturally home to sites of injury, this finding has important clinical implications for the use of MSCs as therapeutics.

58 Holmberg J, Genander M, Halford MM et al.

EphB receptors coordinate migration and proliferation in the intestinal stem cell niche. Cell 125(6), 1151–1163 (2006). 59 Genander M, Halford MM, Xu NJ et al.

Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression. Cell 139(4), 679–692 (2009). 60 Hafner C, Meyer S, Hagen I et al. Ephrin-B

reverse signaling induces expression of wound healing associated genes in IEC-6 intestinal epithelial cells. World J. Gastroenterol. 11(29), 4511–4518 (2005). 61 Hafner C, Meyer S, Langmann T et al.

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn’s disease and contributes to accelerated epithelial wound healing in vitro. World J. Gastroenterol. 11(26), 4024–4031 (2005).

642

n

First to define a subset of MSCs with an intrinsic ability to migrate to the intestine at high efficiency and to integrate into the native tissue.

64 Koo BK, Lim HS, Chang HJ et al. Notch

signaling promotes the generation of EphrinB1-positive intestinal epithelial cells. Gastroenterology 137(1), 145–155, 155.e1–3 (2009). 65 Ingram DA, Mead LE, Tanaka H et al.

Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104(9), 2752–2760 (2004). 66 Wood JA, Colletti E, Mead LE et al. Distinct

contribution of human cord blood-derived endothelial colony forming cells to liver and gut in a fetal sheep model. Hepatology 56(3), 1086–1096 (2012). 67 Powell DW, Adegboyega PA, Di Mari JF,

Mifflin RC. Epithelial cells and their neighbors I. Role of intestinal myofibroblasts in development, repair, and cancer. Am. J. Physiol. Gastrointest. Liver Physiol. 289(1), G2–G7 (2005). 68 Lee RH, Seo MJ, Reger RL et al. Multipotent

stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/SCID mice. Proc. Natl Acad. Sci. USA 103(46), 17438–17443 (2006). 69 Li Y, Chen J, Chen XG et al. Human marrow

stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology 59(4), 514–523 (2002). 70 Togel F, Hu Z, Weiss K, Isaac J, Lange C,

Westenfelder C. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am. J. Physiol. Renal Physiol. 289(1), F31–F42 (2005). 71 Wu GD, Nolta JA, Jin YS et al. Migration of

mesenchymal stem cells to heart allografts during chronic rejection. Transplantation 75(5), 679–685 (2003). 72 Chamberlain J, Yamagami T, Colletti E et al.

Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep. Hepatology 46(6), 1935–1945 (2007). Regen. Med. (2013) 8(5)

73 Colletti EJ, Airey JA, Liu W et al. Generation

of tissue-specific cells from MSC does not require fusion or donor-to-host mitochondrial/membrane transfer. Stem Cell Res. 2(2), 125–138 (2009). 74 Aggarwal S, Pittenger MF. Human

mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105(4), 1815–1822 (2005). 75 Rasmusson I, Ringden O, Sundberg B, Le

Blanc K. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp. Cell Res. 305(1), 33–41 (2005). 76 Corcione A, Benvenuto F, Ferretti E et al.

Human mesenchymal stem cells modulate B-cell functions. Blood 107(1), 367–372 (2006). 77 Di Nicola M, Carlo-Stella C, Magni M et al.

Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99(10), 3838–3843 (2002). 78 Klyushnenkova E, Mosca JD, Zernetkina V

et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J. Biomed. Sci. 12(1), 47–57 (2005). 79 Ramasamy R, Fazekasova H, Lam EW, Soeiro

I, Lombardi G, Dazzi F. Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle. Transplantation 83(1), 71–76 (2007). 80 Spaggiari GM, Capobianco A, Becchetti S,

Mingari MC, Moretta L. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL‑2induced NK‑cell proliferation. Blood 107(4), 1484–1490 (2006). 81 Yang SH, Park MJ, Yoon IH et al. Soluble

mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL‑10. Exp. Mol. Med. 41(5), 315–324 (2009). 82 Paczesny S, Choi SW, Ferrara JL. Acute graft-

versus-host disease: new treatment strategies. Curr. Opin. Hematol. 16(6), 427–436 (2009). 83 Ding Y, Bushell A, Wood KJ. Mesenchymal

stem-cell immunosuppressive capabilities: therapeutic implications in islet transplantation. Transplantation 89(3), 270–273 (2010). 84 Bartholomew A, Sturgeon C, Siatskas M et al.

Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp. Hematol. 30(1), 42–48 (2002). 85 Yagi H, Soto-Gutierrez A, Parekkadan B et al.

Mesenchymal stem cells: mechanisms of immunomodulation and homing. Cell Transplant. 19(6), 667–679 (2010).



86 Devine SM, Cobbs C, Jennings M,

Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. Blood 101(8), 2999–3001 (2003).

T cells, Th17 effector cells, and cytokine environment in inflammatory bowel disease. J. Clin. Immunol. 30(1), 80–89 (2010).

Zetterberg E, Ringden O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp. Hematol. 31(10), 890–896 (2003). 88 Badillo AT, Beggs KJ, Javazon EH, Tebbets

JC, Flake AW. Murine bone marrow stromal progenitor cells elicit an in vivo cellular and humoral alloimmune response. Biol. Blood Marrow Transplant. 13(4), 412–422 (2007). 89 Camp DM, Loeffler DA, Farrah DM,

Borneman JN, Lewitt PA. Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson’s disease. J. Neuroinflammation 6, 17 (2009). 90 Eliopoulos N, Stagg J, Lejeune L, Pommey S,

Galipeau J. Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice. Blood 106(13), 4057–4065 (2005). 91 Nauta AJ, Westerhuis G, Kruisselbrink AB,

Lurvink EG, Willemze R, Fibbe WE. Donorderived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 108(6), 2114–2120 (2006). n

One of the first papers to overturn the widely held dogma that MSCs are invisible to the immune system and can thus be transplanted without regard to allogeneic barriers.

92 Poncelet AJ, Vercruysse J, Saliez A, Gianello

P. Although pig allogeneic mesenchymal stem cells are not immunogenic in vitro, intracardiac injection elicits an immune response in vivo. Transplantation 83(6), 783–790 (2007). 93 Petersdorf EW, Longton GM, Anasetti C

et al. Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors. Blood 89(5), 1818–1823 (1997). 94 Isakova IA, Dufour J, Lanclos C, Bruhn J,

Phinney DG. Cell-dose-dependent increases in circulating levels of immune effector cells in rhesus macaques following intracranial injection of allogeneic MSCs. Exp. Hematol. 38(10), 957–967.e1 (2010). 95 Yadav PK, Chen C, Liu Z. Potential role of

NK cells in the pathogenesis of inflammatory bowel disease. J. Biomed. Biotechnol. 2011, 348530 (2011). 96 Eastaff-Leung N, Mabarrack N, Barbour A,

Cummins A, Barry S. Foxp3+ regulatory


on cytomegalovirus. Antiviral Res. 39(3), 141–162 (1998). 108 Gallant JE, Moore RD, Richman DD, Keruly

97 Kanai T, Kawamura T, Dohi T et al.

J, Chaisson RE. Incidence and natural history of cytomegalovirus disease in patients with advanced human immunodeficiency virus disease treated with zidovudine. The Zidovudine Epidemiology Study Group. J. Infect. Dis. 166(6), 1223–1227 (1992).

Th1/Th2-mediated colitis induced by adoptive transfer of CD4+CD45RBhigh T lymphocytes into nude mice. Inflamm. Bowel Dis. 12(2), 89–99 (2006).

87 Le Blanc K, Tammik C, Rosendahl K,

n

Describes the induction of an immunemediated, inflammatory bowel disease-like syndrome in mice, providing a valuable preclinical model system in which to test putative therapies.

98 Maloy KJ. Induction and regulation of

inflammatory bowel disease in immunodeficient mice by distinct CD4+ T-cell subsets. Methods Mol. Biol. 380, 327–335 (2007).

109 Meyers JD, Flournoy N, Thomas ED. Risk

factors for cytomegalovirus infection after human marrow transplantation. J. Infect. Dis. 153(3), 478–488 (1986). 110 Lin A, Xu H, Yan W. Modulation of HLA

expression in human cytomegalovirus immune evasion. Cell. Mol. Immunol. 4(2), 91–98 (2007). 111 Ploegh HL. Viral strategies of immune

evasion. Science 280(5361), 248–253 (1998).

99 Ostanin DV, Bao J, Koboziev I et al. T cell

transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am. J. Physiol. Gastrointest. Liver Physiol. 296(2), G135–G146 (2009). 100 Sturm A, de Souza HS, Fiocchi C. Mucosal

112 Tortorella D, Gewurz BE, Furman MH,

Schust DJ, Ploegh HL. Viral subversion of the immune system. Annu. Rev. Immunol. 18, 861–926 (2000). 113 Soland MA, Bego MG, Colletti E et al.

T cell proliferation and apoptosis in inflammatory bowel disease. Curr. Drug Targets 9(5), 381–387 (2008).

Modulation of human mesenchymal stem cell immunogenicity through forced expression of human cytomegalovirus US proteins. PLoS ONE 7(5), e36163 (2012).

101 Kudo K, Liu Y, Takahashi K et al.

Transplantation of mesenchymal stem cells to prevent radiation-induced intestinal injury in mice. J. Radiat. Res. (Tokyo) 51(1), 73–79 (2010). 102 Hayashi Y, Tsuji S, Tsujii M et al. The

transdifferentiation of bone-marrow-derived cells in colonic mucosal regeneration after dextran-sulfate-sodium-induced colitis in mice. Pharmacology 80(4), 193–199 (2007). 103 Yabana T, Arimura Y, Tanaka H et al.

Enhancing epithelial engraftment of rat mesenchymal stem cells restores epithelial barrier integrity. J. Pathol. 218(3), 350–359 (2009). 104 Gonzalez MA, Gonzalez-Rey E, Rico L,

Buscher D, Delgado M. Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology 136(3), 978–989 (2009). 105 Mouiseddine M, Francois S, Semont A et al.

Human mesenchymal stem cells home specifically to radiation-injured tissues in a non-obese diabetes/severe combined immunodeficiency mouse model. Br. J. Radiol. 80(Spec. No. 1), S49–S55 (2007). 106 Bilgrami S, Almeida GD, Quinn JJ et al.

Pancytopenia in allogeneic marrow transplant recipients: role of cytomegalovirus. Br. J. Haematol. 87(2), 357–362 (1994). 107 De Jong MD, Galasso GJ, Gazzard B et al.

Summary of the II International Symposium

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First paper to show that the immunogenicity of MSCs could be markedly reduced by expressing proteins that human cytomegalovirus uses to evade the host immune system.

114 Colletti E, Airey JA, Liu W et al. Generation

of tissue-specific cells by MSC does not require fusion or donor to host mitochondrial/membrane transfer. Stem Cell Res. 2, 125–138 (2009). 115 Jones DR. Why do in utero stem cell

transplants sometimes fail? Mol. Med. Today 5(7), 288–291 (1999). 116 Nauta AJ, Fibbe WE. Immunomodulatory

properties of mesenchymal stromal cells. Blood 110(10), 3499–3506 (2007). 117 Medzhitov R, Janeway CA. Decoding the

patterns of self and nonself by the innate immune system. Science 296(5566), 298–300 (2002). 118 Colvin RB, Smith RN. Antibody-mediated

organ-allograft rejection. Nat. Rev. Immunol. 5(10), 807–817 (2005). 119 Sacks SH, Zhou W. Allograft rejection: effect

of local synthesis of complement. Springer Semin. Immunopathol. 27(3), 332–344 (2005). 120 Wasowska BA, Lee CY, Halushka MK,

Baldwin WM 3rd. New concepts of complement in allorecognition and graft rejection. Cell. Immunol. 248(1), 18–30 (2007).

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Review


121 Dunkelberger JR, Song WC. Complement

and its role in innate and adaptive immune responses. Cell Res. 20(1), 34–50 (2010). 122 Sacks SH, Chowdhury P, Zhou W. Role of

the complement system in rejection. Curr. Opin. Immunol. 15(5), 487–492 (2003). 123 Schraufstatter IU, Discipio RG, Zhao M,

Khaldoyanidi SK. C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation. J. Immunol. 182(6), 3827–3836 (2009). 124 Moll G, Jitschin R, von Bahr L et al.

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses. PLoS ONE 6(7), e21703 (2011). 125 Ignatius A, Schoengraf P, Kreja L et al.

Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL‑1b. J. Cell. Biochem. 112(9), 2594–2605 (2011). 126 Tu Z, Li Q, Bu H, Lin F. Mesenchymal stem

cells inhibit complement activation by secreting factor H. Stem Cells Dev. 19(11), 1803–1809 (2010).

with serum. Blood 120(17), 3436–3443 (2012). n

Extends prior work showing that MSCs are not invisible to the immune system by demonstrating that they are recognized by components of the innate immune system, thus identifying another obstacle that needs to be overcome when using MSCs as therapeutics.

128 Chen G, Yang Y, Gao X et al. Blockade of

complement activation product C5a activity using specific antibody attenuates intestinal damage in trinitrobenzene sulfonic acid induced model of colitis. Lab. Invest. 91(3), 472–483 (2011). 129 Jain U, Woodruff TM, Stadnyk AW. The C5a

receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL‑4 and IL‑10. Br. J. Pharmacol. 168(2), 488–501 (2013). 130 Soland MA, Bego M, Colletti E et al.

Mesenchymal stem cells engineered to inhibit complement-mediated damage. PLoS ONE 8(3), e60461 (2013). 131 Spear GT, Lurain NS, Parker CJ, Ghassemi

M, Payne GH, Saifuddin M. Host cell-derived complement control proteins CD55 and

CD59 are incorporated into the virions of two unrelated enveloped viruses. Human T cell leukemia/lymphoma virus type I (HTLV-I) and human cytomegalovirus (HCMV). J. Immunol. 155(9), 4376–4381 (1995). 132 Spiller OB, Morgan BP, Tufaro F, Devine DV.

Altered expression of host-encoded complement regulators on human cytomegalovirus-infected cells. Eur. J. Immunol. 26(7), 1532–1538 (1996). 133 de la Garza-Rodea AS, Verweij MC, Boersma

H et al. Exploitation of herpesvirus immune evasion strategies to modify the immunogenicity of human mesenchymal stem cell transplants. PLoS ONE 6(1), e14493 (2011). 134 Kim JY, Kim D, Choi I et al. MHC

expression in a human adult stem cell line and its down-regulation by HCMV US gene transfection. Int. J. Biochem. Cell. Biol. 37(1), 69–78 (2005). 135 Lee EM, Kim JY, Cho BR et al. Down-

regulation of MHC class I expression in human neuronal stem cells using viral stealth mechanism. Biochem. Biophys. Res. Commun. 326(4), 825–835 (2005).

127 Li Y, Lin F. Mesenchymal stem cells are

injured by complement after their contact

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