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Stem cells therapies for gastrointestinal and liver diseases
R. Iacob, P. Sîrbu-Boeti, S. Iacob, S. Dima, C. Gheorghe, L. Gheorghe, I. Popescu (Chirurgia, 104 (2): 131-140)

Stem cells - definitions and key biological properties
Stem cells research represents a new field of biomedical science on which scientists have focused their attention during the last two decades and which is rapidly developing into a new cutting edge area of research, offering many new opportunities for novel therapeutic approaches to diseases until now hopelessly incurable.
It is known that stem cells have two fundamental properties. Self renewal - represents the ability to preserve its own cell population. The process called symmetrical division represents the mitosis of the stem cell generating two identical daughter cells which could be stem or differentiating elements. The asymmetrical division produces one stem cell and one cell that leaves the stem cell compartment in order to start a certain differentiation process. The second fundamental stem cell property is the multipotency that is the capacity to generate multiple linage progenitors by differentiation, a progressive cellular phenotype modification due to dynamic changes in gene expression pathways.
Embryonic stem cells (ESCs) have the capacity of indefinite self replication and self renewal and, at the same time, they are pluripotent, meaning that, under appropriate conditions, they can differentiate into every cell lineage that makes up the mature organism. In mammals, such stem cells constitute the inner cell mass of the blastocyst. As the potential therapeutic use of human embryonic stem cells has always been counterbalanced by ethical and legal aspects (1,2), recent discoveries have reoriented stem cell research also to other sources of stem cells.
The first of these discoveries was that pluripotent stem cells are also present in most organs of the mature adult body and are generating the progenitor cells for the periodic cell turnover of the organs' tissues, being used for repair or replacement of cells that have been injured, destroyed or undergone apoptosis (3). The transition from totipotent embryonic stem cells to pluripotent organ-based stem cells is not yet understood and it is unknown whether in the transition process, the embryonic stem cells are losing some of their differentiation potential. But the organ-based adult stem cells certainly retain their full ability of self-replication which is an essential component of being a stem cell. Among the various organ-based stem cells, the hematopoietic stem cells of the bone-marrow are the most studied and well characterized (4-6).
Currently two types of stem cells have been recognized: true stem cells, fewer in number but with a greater differentiation potential and a longer proliferative capacity and short term elements, that are believed to derive from the first ones as committed progenitors, able to differentiate into mature elements to provide tissue specific functions (7). The tissue specific stem cells play an important role in the maintenance of tissue homeostasis participating in tissue turnover and providing tissue regeneration in case of cell damage.
The source of stem cells is largely debated: one hypothesis states that they can derive from the progressive specialization of ESCs that generate each tissue but they could also appear later in the development from circulating stem cells engrafted in all organs to participate to their regeneration and renewal (8). Conventional belief denied the possibility of reprogramming the adult stem cells but several recent reports introduced the term transdifferentiation, representing the ability of stem cells from one tissue to change into another, even of different dermal origin (8). It appears to be a matter of genetic reprogramming as all the information is contained in the same sets of genes universally available, but activated for expression according to different cell programs. Genetically labeled neural stem cells - neuroectoderm can give rise to a variety of blood cells (mesoderm) (9); Stem cells isolated from muscle are able to differentiate into blood cells after transplantation to an irradiated receptor (10); in patients receiving transplantation of hematopoietic stem cells (HSC) from peripheral blood or bone marrow after chemo-radiotherapy for cancer donor-derived differentiated cells were identified in biopsies of the liver, gastrointestinal mucosa or skin, suggesting that HSCs can generate by transdifferention hepatocytes and epithelial cells (endoderm and ectoderm) (11). All these evidence suggest that tissue-specific stem cells have an important degree of developmental and differentiation plasticity in which a key role is played by the environment around the cells representing a sort of permissive niche able to determine the cell fate: quiescence, proliferation, apoptosis or differentiation. A large panel of signaling molecules as well as of cell-matrix interactions is important in determining stem cell fate for homeostasis maintenance through negative feedback control process.
A particular degree of plasticity and heterogeneity among adult stem cells is shown by HSCs, the cellular elements that permanently regenerate the blood forming system (12). The membrane glycoprotein CD34 has been shown to be a usefull marker of HSCs and progenitor cells (13). The clinical applications of HSCs began in 1963 with the first cases of bone marrow transplantation performed in order to permit the eradication of endogenous cancers increasing chemotherapy and radiotherapy to myeloablative doses. HSC are the multipotent elements from which all the components of the blood generate, according to the monophyletic theory (Maximov 1909) (14). Until 1950 it was thought that no immature cells were present in the blood streams, but in 1962 Gordman et al. demonstrated that stem cells circulate normally in the peripheral blood opening the door to speculations concerning the primary role of these itinerant elements for embryonic development of hematopoiesis and lymphopoiesis in the appropriate organ and for maintenance of the blood compartment in the adulthood (15). In vitro culture and in vivo transplantation assays have demonstrated the great plasticity of HSCs able to give rise to a wide array of phenotypes including blood, cartilage, fat, tendon, lung, liver, muscle, marrow, stroma, brain (16), heart (17) and kidney cells (18). HSCs may represent multipotent multisomatic stem cells which provide an alternative source of stem cells for all tissues, localizing into any organ under conditions of stress and providing for its regeneration or perhaps becoming, even in rest conditions a dormant tissue specific stem cell able to participate in tissue renewal (8).

Hepatic and gastrointestinal stem cells
Despite many efforts made during the last 20 years the knowledge acquired on gastrointestinal and hepatic stem cells is still incomplete and sometimes contradictory, many questions being yet to be solved: do intestinal and liver stem cells really exist, what are their molecular and phenotypic characteristics, what is their origin and their cell fate, under what mechanisms proliferate and differentiate, what is their role in homeostasis of the gastrointestinal tract and in disease pathogenesis or do they have a role in prevention or cure of gastrointestinal disease? (19)
Recent studies indicate that in the liver, multipotent hepatic stem cells might be found in the Canal of Hering (20,21) a ductular structure that is the link between the hepatocyte's canalicular system and the biliary tree. Proliferating liver stem cells generate oval cells which are progenitors of mature hepatocytes and biliary epithelial cells, cells with a very low turnover compared to cells of the bone marrow or intestinal mucosa. Usually, in the liver, replacement of physiologically aging hepatocytes, or of mature cells damaged by infections or toxins, primarily occurs by proliferation of viable hepatocytes in situ. Hepatic stem cells are activated only in case of a very severe liver damage, that destroys or blocks most resident hepatocytes to enter the cell growth cycle (22). Only under these conditions oval cells can be identified in the liver (23).
Important findings have been provided by studies conducted by Sell et al. who have demonstrated that, in animal models, there are three levels of proliferating cells in the liver, differently recruited according to the extent of damage, its localization and nature (7,24): the mature hepatocytes, the ductular bipolar progenitor cells and last but not least, periductular putative liver stem cells.
The proliferative response generated in the liver by different types of injuries is called ductular reaction and is characterized by an increase in duct-like structures. It has been classified in typical and atypical (cholangiolar and oval cell proliferation) depending on the predominant appearance of proliferating cells (25). A typical ductular reaction can be observed even in the case of an acute extrahepatic cholestasis and is similar to the proliferation of the mature well formed ducts. By contrast the proliferation of bile ductules located adjacent to the parenchyma and arranged into anastomosing chords defines an atypical reaction. This type of ductular reaction might be observed in several hepatic disorders such as chronic intrahepatic or extrahepatic cholestasis, sub-massive necrosis, alcoholic liver disease, focal nodular hyperplasia or liver allograft failure. In case of massive and sub-massive hepatocellular necrosis, active liver cirrhosis, chronic active hepatitis and severe alcoholic liver disease oval cell proliferation, a subtype of atypical ductular reaction has been identified and it has been described for the first time by Opie in 1944. The oval cells are small cells with oval nuclei that can be identified in rat livers after exposure to certain carcinogenic injury, initially around the portal space and then invading the whole hepatic lobule (26). Currently the term oval cells is used to define the emergence in both human and animal livers of small size proliferating elements with a voluminous nucleus, that occur after sublethal massive liver necrosis induced by hypoxia or chemicals (27). Due to their high proliferative capacity and the ability to differentiate both in vivo and in vitro into hepatocytes and billiary cells, oval cells are considered bipotential liver stem cells (28).
The mucosal cells which line the small crypts of the intestine and villi, in contrast to hepatocytes, exhibit a very rapid physiological turnover, by migrating in 3-4 days from the base of the crypt where they originate, to the upper part of the villous structure where they eventually are shed into the intestinal lumen. Very few intestinal stem cells are located in each crypt's base where they predominantly divide into one self-replicated stem cell and one progenitor daughter cell through an asymmetrical division process. The latter, while moving up along the inside wall of the crypt, undergoes a series of divisions and acquires differential maturity when reaching the base of the villus generating at least four major cell lineages: columnar cells, mucin producing goblet cells, Paneth cells and, probably, selected endocrine cells. There is also data to support the latter origin from neural stem cells (29). One might conclude that in contrast to hepatic stem cells, the primary function of intestinal stem cells appears to be the provision of progenitors for replacement of the rapidly turning-over mucosal cells.
Animal models of gut injury have been used in order to assess the ability of stem cells to regenerate damaged tissues. Using the microcolony clonogenic stem cell assay it has been showed that in the murine bowel the regenerating crypts after toxic injury (radiation or cytotoxic treatment) appear over the course of 3 days. The extent and localization of recruitment in the clonogenic compartment is determined by the level of damage. Such studies have suggested that a hierarchy of stem cells organized in 3 main categories exists, having different degree of injury tolerance and which are progressively recruited in order to achieve crypt survival. Exposed to low doses of radiation, true stem cells appear to be extremely sensitive to DNA damage and unable to repair themselves; when this compartment is completely destroyed (by a p53 mediated apoptosis mechanism) a second level of multipotent stem cells emerge, having an intermediate radio-tolerance and which express their stem potential under stress conditions and are able to regenerate the destroyed first compartment. Finally higher doses of injury seem to involve a third level highly injury tolerant clonogenic population, possibly the latest generation of progenitors with stemness potential (30).

Stem cells therapies in hepatology
The interest in alternative methods of liver support like liver cell therapies has increased recently, as the demand of whole organs for liver transplantation far outweights the organ supply and the waiting lists for liver transplantation are constantly increasing. There are interesting reports that have already shown that cell based transplantation protocols can be used as a bridge to liver transplant (31,32) and can be used repeatedly, to decrease mortality in acute liver failure (33,34) or to treat end stage liver metabolic disease (35-38). The development of these techniques is dependant on the availability of adequate number of hepatocytes for transplantation and there are already at least two possible sources to take into account: stem and precursor cells to generate hepatocyte-like cells or reversibly replicated hepatocyte cell lines (39). In recent reports 'hepatocyte-like cells' have been generated from different types of extrahepatic stem or precursor cells.
During the last 8-9 years there have been many reports of hepatocyte like cells generated from different types of extrahepatic stem or precursor cells. However, before entering on a large scale in a human clinical trial setting, more basic research has to be concluded. On the other hand, the characterization of stem cell biology in animal work is clearly important, but clinical application requires convincing evidence that human stem cells also share the properties demonstrated by adult rodent stem cells. Cellular phenotypes of human hepatic stem cells and tissue reactions similar to those seen in animal models have been described in a variety of human acute and chronic liver diseases (40,41).
It has already been shown that hematopoietic stem cells were capable of differentiating into hepatocytes and cholangiocytes yielding in a high degree of engraftment within injured rodent livers (42). Different markers are tested in order to show the hepatocyte differentiation: albumin, alphafetoprotein, GATA4, HepPar1 antigen, CK19, glutamine synthetase, transferrine (43-46). When human precursor cells were used in order to obtain hepatocytes before transplantation in an animal model, usually single cells or small clusters of hepatocyte-like cells were observed in the animal liver after transplantation. An improvement of liver function in mouse models by cell therapy with human extrahepatic stem cells has not yet been consistently reported. Best results, with more than 20% albumin-producing human parenchymal hepatic cells, have been presented after transplantation of adherently proliferating cord blood cells into fetal sheep (47), but these results have to be independently confirmed. The transplantation of rodent stem cells into rodents yields better results, as the restoration of liver function after stem cells transplantation into CCl4 pretreated mice has been reported after 2-7 days after transplantation (48). Important future studies should focus on transplantation of human stem cells using pig liver disease models as the pig liver may represent a more realistic model with regard to human liver regeneration and also resemble more closely to the human setting with regard to size and anatomical structure (49).
Based on the available data it is difficult to compare the different human cell types with respect of their capacity to differentiate to hepatocytes in vivo. Various cell types have been studied so far: adherently proliferating cells from human cord blood, CD34+ mononuclear cell fraction from cord blood, haematopoietic cells from bone marrow, nestin-positive pancreatic islet cells, hepatocyte-like cells isolated from peripheral blood monocytes by a two-step dedifferentiation/differentiation in vitro protocol, amniotic epithelial cells that develop from the epiblast by 8 days after fertilization. There are advantages and drawbacks for each cell type: an advantage of the adherently proliferating cells is the availability of large amounts of cells whereas an advantage of the monocyte derived hepatocyte-like cells (50) is the opportunity to generate these cells from the recipient's own blood, thus avoiding immune suppressive medication. Especially for the proliferating cell types it is crucial to exclude malignant transformation after transplantation.
The mechanism underlying the generation of hepatocytes after stem cell transplantation is still a matter of important debate and at least two hypotheses are discussed: the transdifferentiation pathway, in which under the influence of factors of the host's microenvironement the transplanted stem cells achieve a hepatocyte differentiation and the fusogenic theory in which stem cells fuse with adult host cells and undergo a genetic reprogramming sequence. This last mechanism could be of paramount importance in delivering wild type genes to deficient host liver cells thus correcting genetic defects of these cells especially in metabolic liver disease. However, it should be taken into consideration that fusogenic therapy could have also deleterious consequences as cell fusion leads to aneuploidy and eventually also to chromosome instability and loss of chromosomes. Neoplasia must be carefully ruled out before fusogenic therapy can be applied in a clinical setting.
Data from mature hepatocyte transplantation studies have indicated that the main therapeutic classes that can benefit from cell based therapies in liver disease are: acute liver failure, inherited metabolic liver disease and end-stage liver disease (cirrhosis) (49).
In acute liver failure due to viral hepatitis, idiosyncratic drug reactions, acetaminophen or mushroom ingestion, cell therapies should provide rapid liver support improving hepatic encephalopathy, brain edema, coagulopathy, multiorgan failure as it has already been shown for transplant of allogeneic primary hepatocytes isolated from cadaver livers (51). There are different routes of hepatocyte administration: intraperitoneal administration with or without encapsulation or attachment to micro-carriers, splenic artery or portal vein infusion (52). However the evaluation of cell therapies in acute liver failure may be difficult due to large variations in the course of the disease, multiple aetiologies, complex supportive treatment and the important spontaneous recovery rate of approximately 20% by liver self-regeneration.
Metabolic disease are possibly better candidates for cell transplantation as the course of metabolic liver disease usually varies less and objective parameters such as laboratory data can be determined to assess the efficacy of the treatment but one should take into account that these conditions are rarely immediately life threatening and often acceptable conventional therapies are available. Therefore the potential benefits must be carefully weighted against any possible complications after cell therapies, such as immunosuppression, hepatocyte embolisation of the pulmonary vascular system, sepsis or haemodynamic instability. Several case reports have already been published presenting positive results of hepatocyte transplantation for different metabolic disease (53-56). Significant aspects are related to long term engraftment and whether or not transplanted cells get a selection advantage over the receptors hepatocytes, especially in case of metabolic disease in which hepatocyte damage occurs (e.g. Wilson's Disease), leading to major improvement in the disease course.
More studies are needed on the potential use of stem cells transplantation in metabolic disease. Allogenic transplantation of mesenchymal stem cells or of hepatocyte precursors obtained from stem cells might be of great interest in the treatment of metabolic end-stage liver diseases. After transplantation, these cells might represent a cellular population without genetic defect and have the potential even to cure the metabolic disease (e.g. Wilsons Disease) if the transplanted cells could get a selection advantage in comparison with the hosts hepatocytes.
Cell transplantation for end-stage cirrhosis brings additional problems related to loss of functional hepatocytes, abnormalities of hepatic architecture and intra-hepatic porto-portal shunts, the benefit of additionally transplanted hepatocytes into the liver without restoring the normal liver architecture being questionable. When the liver architecture is deranged, cell infusions may cause prolonged portal hypertension and embolization in the lung (57). In this setting, different extrahepatic engraftment sites could be taken into account and the best studied ectopic site is the spleen.
Other routes of administration are infusion in the portal or mesenteric veins, direct intrasplenic or intrahepatic injection, kidney subcapsular injection or intraperitoneal delivery of encapsulated hepatocytes or of hepatocytes attached to microspheres.
Related to the number of transplanted hepatocytes that can induce a therapeutic benefit it is considered that between 1.8-8.8x109 transplanted hepatocytes can be of clinical significance (1-5% of liver cell mass).
It has been shown that mesenchymal stem cells (MSCs) from adult bone marrow preferentially home to damaged tissue and may have therapeutic potential as they can differentiate in vitro and in vivo into various cell types, such as bone, fat and cartilage or hepatocytes. In vitro data suggest that MSCs have low inherent immunogenicity as they induce little, if any, proliferation of allogenic lymphocytes and even appear to be immunosuppressive in vitro. They inhibit T-cell proliferation to alloantigens and mitogens and prevent the development of cytotoxic T-cells. Possible clinical applications include therapy-resistant severe acute graft-versus-host disease, tissue repair, treatment of rejection of organ allografts and autoimmune disorders. Future studies on cell therapies in liver diseases might be focused on achieving immunosuppressive free liver transplantation using concomitant mesenchymal stem cells transplantation or on the treatment of autoimmune liver disease with mesenchymal stem cells transplantation in order to obtain long term remission of the disease.

Stem cells therapies for gastrointestinal diseases
One of the most important research fields in gastro-enterology nowadays is represented by inflammatory bowel diseases (IBD). There are mainly two clinical entities recognized - Crohn's Disease (CD) and ulcerative colitis (UC), which are characterised by a chronic recurrent inflammation of the gastrointestinal tract with significant genetic predisposition. They are diagnosed mainly in young patients during the second or the third decade of life and, especially for CD, have a major negative impact on patients quality of life. The interactions between the genetic background of predisposed individuals and environmental factors that trigger the onset of IBD is yet incompletely elucidated but a significant role seems to be played by the loss of immune tolerance of gastrointestinal tract to environmental antigens, with impaired activation of immune system and TH1/TH2 imbalance (58,59).
As hematopoietic stem cell transplantation has already been shown to be beneficial in diseases characterised by an over activation of TH1 immune response, the use of HSC for IBD therapy has been proposed, especially for CD, in which a predominantly TH1 immune response has been shown. In this setting, allogenic bone marrow transplantation in patients with CD might be beneficial as it may correct the genetic abnormalities present in circulating leucocytes. Autologous bone marrow transplantation might also be beneficial, as it may reset the host immune system, restoring the status before the onset of IBD, when the organism was only predisposed for developing the disease but the disease was not yet active. A potential mechanism of action is the elimination of immunological active cell populations. In other diseases the resetting of immune system after transplantation might last for months or years and in some cases the re-emergence of initial cellular populations might never occur with the chance of a long lasting remission.
An alternative explanation for efficacy of stem cell therapies in IBD is represented by the fact that autologous HSC transplantation permits the use of much more aggressive immunosuppressive regimens. As immunosupresive therapy is the base of IBD therapy the use of more aggressive regiments has the potential to generate a fundamental immunological "switch", providing long term remission.
Initial reports on efficacy of stem cell therapies in IBD have been presented for patients with IBD receiving HSC transplantation usually for haematological diseases (60). In 1993 a first CD case in which allogenic bone marrow transplantation has been performed for a lymphoma has been reported (61). Post-transplantation an improvement in IBD has been noted, although follow-up was extended only for 6 months after transplantation. Of particular interest was the report in 1998 of a series of 5 CD patients with favourable evolution after allogenic bone marrow transplantation, strongly suggesting the possible use of this therapeutic approach in selected cases. Among 5 patients with active IBD in the pretransplant setting, a more than 10 years remission was recorded for 3 patients, despite the discontinuation of immunosuppressive therapy. One patient, however, had a relapse after 8 months, with the need for a surgical intervention thereafter. These data have strongly suggested the potential benefit of stem cell therapies in IBD but also have shown that the benefit may not be universal (62).
In 2003 long term follow-up (median 10 years, 0.5-22 years) after bone marrow transplantation in IBD patients has been reported for 11 patients, 7 patients with CD and 4 with UC. Six of the 11 patients had active IBD at the moment of medular conditioning, receiving sulfasalazine or corticosteroid therapy. In 10 of the 11 patients long term IBD remission has been recorded after allogenic stem cell transplantation (63).
There have been reported cases with new onset of IBD after bone marrow transplantation and genetic testing has revealed the presence of NOD2 mutations in the transplanted cells population, supporting the genetic base of IBD etiopathogeny (64).
Several studies have reported the evolution of IBD patients after autologous stem HSCs transplantation for different pathologies. In one patient diagnosed with IBD at age of 13 and treated for IBD for 7 years who underwent an autologous HSC transplantation for non-Hodgkin lymphoma, a 7 years remission of IBD has been recorded after stem cells transplantation (65). A 5 years remission of IBD has been documented for another patient diagnosed with IBD 2 years before HSC transplantation (66). In patients with UC both induction of remission and flare-ups after stem cells transplantation have been recorded.
The first IBD series in which stem cells transplantation has been specifically performed for IBD has been presented by a research group from Chicago, in patients with active CD with CDAI >250, non responsive to biological therapies (67). Stem cells mobilization has been performed with cyclophosphamide and granulocyte-colony stimulating factor (G-CSF), followed by ex-vivo CD34+ selection. Before retransplantation immune conditioning has been performed using cyclophosphamide and anti-thymocite globulin. A significant clinical and colonoscopic improvement of IBD lesions has been registered after HSCs transplantation.
The first phase I clinical trial on HSCs transplantation for refractory CD has been published by Oyama et al in 2005. The study has involved 12 patients with CDAI of 250-400 despite conventional treatments. Stem cells transplantation has been tolerated by all patients with usual adverse events like fever, diarrhea, nausea, vomiting. The median days for neutrophil and platelet engraftment were 9.5 (range, 8-11) and 9 (range, 9-18), respectively. Eleven of the 12 patients have presented a clinical remission with a CDAI <150, for a median follow-up of 18.5 months. In one patient a recurrence of CD after 15 months posttransplantation has been recorded (68).
Recent data has been published by an Italian group on the efficacy of HSC transplantation with unselected peripheral blood stem cells (PBSC) in moderate-severe refractory CD. The study has involved 4 CD patients with a mean CDAI of 319, intolerant or non-responsive to multiple conventional therapeutic regimens. Unselected PBSC were collected after mobilisation with cyclophosphamide 1.5 g/m2 and G-CSF 10 mg/kg. Three months after transplantation all 4 patients were in clinical remission with a CDAI score of 91 and complete endoscopic remission has been found in 2 patients. After a median follow-up of 16.5 months clinical remission and fistulas closure has been registered for 3 out of 4 patients, with no additional treatment (69).
These positive data are encouraging, taking into account the severity of treated patients and the long term remission documented posttranplantation and justify randomized, controlled, phase II trials. An European multicentric trial for evaluation of HSCs transplantation in CD is ongoing and coordinated by Prof. C. Hawkey (UK) and it aims to compare two HSCs transplantation regimens: stem cells transplantation immediately after mobilisation vs. transplantation 1 year after mobilization. This study design will be able to verify the therapeutic potential of all components of the transplantation regimen as the mobilization procedure per-se might have therapeutic benefit, distinct from the transplant procedure (70).
Intestinal fistulas continue to pose significant problems in gastrointestinal tract surgery, despite surgical technical progress and the availability of new treatment modalities. As surgical procedures have become more complex and more radical, the incidence and severity of postoperatory fistulas have increased, as well as postoperative mortality. Sadly, these complications have still a high rate of mortality, which might rise over 60%.
Once the mechanisms of tissue healing have started to be understood, an increased interest has been shown to conjunctive tissue, a key player in this process. An important role is played by the extracellular matrix, especially by the collagen fibers which assure tissue mechanical resistance. Moreover recent studies have shown that interactions between cells and extracellular matrix collagen fibers significantly influence cells fate, activating cellular division, cell differentiation and stimulating or inhibiting collagen synthesis. Some in vitro studies have shown that different collagen substrates extracted from conjunctive tissue significantly impact cells morphology and cell proliferation in culture. Cellular response seems to depend on the physical-chemical features of collagen fibres. Multiple collagen cellular interactions are due to the alpha chains in its structure. Cells reactions to collagen depend on collagen hydrophobicity, the water content as well as on surface ionic charge (71).
In the late 80's it has been postulated that tissue regeneration might be achieved using a collagenic structure which contains no nonabsorbable synthetic molecules (72).
It has been shown that the incorporation of collagenic structures (e.g. Alloderm, dura mater umana, elastine patch) to digestive tract leads to digestive regeneration, but the regenerated structure has no or few muscular fibres, which might impair the peristaltic waves and determine the formation of a diverticula. The supplementation of these collagen structures with particular cells might help in regenerating of all the digestive wall layers. Mesenchymal stem cells seeded on collagenic structures have already been successfully used in experimental studies on rats, being capable to determine the regeneration of the four layers of the digestive wall. The potential use of stem cell based therapies in postoperative fistula closure is currently of great interest.
With regard to spontaneous fistulas in the course of IBD the standard of care nowadays is represented by biological anti-TNF agents, with variable success rates. There is research with regard to potential use of mesenchymal stem cells isolated from adipose tissue for IBD fistulas closure and a phase I trial has already been published in 2005. The aim of this study has been to inoculate into the fistula tract autologous mesenchymal stem cells isolated from the adipose tissue in 4 CD patients presenting with 9 complex fistulas. Eight of them have been followed-up constantly for 8 weeks in order to document the fistula closure. In 6 out of 8 fistulas, the outer opening closed after 8 weeks following mesenchymal stem cell injection into the fistula tract and in the other 2 the fistula drainage decreased a lowering in the fistula drainage. No adverse event was noted for a period of 22 months after the procedure (73). The same Spanish group has reported in 2003 the case of one CD patient with recurrent rectovaginal fistula successfully treated by mesenchymal stem cell transplantation using adipose tissue mesenchymal stem cells isolated after liposuction (74).
True Refractory Coeliac Disease is defined as persisting or recurring villous atrophy with crypt hyperplasia and increased intraepithelial lymphocytes in spite of a strict gluten free diet for more than 12 months or when severe persisting symptoms necessitate intervention independent of the duration of the dietary therapy (75). Refractory celiac disease with aberrant T cells (RCD type II) is know to be unresponsive to available therapies while it presents a high risk of transition into enteropathy associated T-cell lymphoma (EATL). In a recent published trial (76) seven patients out of 13 RCD type II patients underwent auto-logous HSCs transplantation, after conditioning with fludarabine and melphalan. All 7 patients completed the mobilization and leukapheresis procedures successfully and subsequently underwent conditioning and transplantation, with engraftment occurring in all patients. There was a significant reduction in the aberrant T cells in duodenal biopsies associated with improvement in clinical well-being and normalization of hematologic and biochemical markers (mean follow-up, 15.5 months; range, 7-30 months) with no major nonhematologic toxicity or transplantation-related mortality observed. These preliminary results showed that high-dose chemotherapy followed by autologous HSCs transplantation seems feasible and safe and might result in long-term improvement of patients with RCD type II.
In conclusion, studies conducted so far have indicated the possibility of successful isolation of stem cells from multiple sources and the successful differentiation of cells on multiple lineages. Preliminary animal and human phase I trials have indicated potential use of stem cells therapies for treatment of yet incurable digestive pathology as end-stage liver disease, inflammatory bowel diseases or refractory celiac disease. More research is still needed for perfecting stem cells harvesting protocols, in vitro expansion and differentiation protocols which can be used in phase II and III human trials.

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