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
The management of perianal fistula in patients with Crohn’s disease is an extremely challenging medical problem as many fistulas do not respond to available treatments. The objectives were to assess the safety and efficacy of a suspension of expanded adipose-derived allogeneic mesenchymal stem cells (eASCs) for the treatment of complex perianal fistula in Crohn’s disease
An open-label, single-arm clinical trial was conducted at six Spanish hospitals. Twenty-four patients were administered intralesionally with 20 million eASCs in one draining fistula tract. A subsequent administration of 40 million eASCs was performed if fistula closure was incomplete at week 12. Subjects were followed until week 24 after the initial administration.
Treatment-related adverse events did not indicate any clinical safety concerns after 6 months follow-up. The full analysis of efficacy data at week 24 showed 69.2 % of the patients with a reduction in the number of draining fistulas, 56.3 % of the patients achieved complete closure of the treated fistula achieved, and 30 % of the cases presenting complete closure of all existing fistula tracts. Of note, closure was strictly defined as: absence of suppuration through the external orifice and complete re-epithelization, plus absence of collections measured by magnetic resonance image scan (MRI). Furthermore, MRI Score of Severity showed statistically significant differences at week 12 with a marked reduction at week 24.
Locally injected eASCs appear to be a simple, safe, and beneficial therapy for perianal fistula in Crohn’s disease patients. Additional studies are needed to further confirm the efficacy of the eASCs.
KeywordsFistulizing Crohn’s disease Perianal fistula Allogeneic adult stem cells
Crohn’s disease is characterized by an uncontrolled immune response to intestinal bacteria causing tissue damage which, in the penetrating phenotype, involves the entire gut wall and results in the formation of fistula tracts . The initial lesion in the perianal area of the Crohn’s patients causes local inflammation and leads to cellular infiltration and secretion of pro-inflammatory cytokines. This immune response is uncontrolled and the tissue remains chronically inflamed preventing fistula healing. Perianal disease is a very disabling chronic condition [2, 3] that is found in approximately 20 % of Crohn’s disease (CD) patients .
The complexity of this condition requires the participation of a multidisciplinary team that includes gastroenterologists, surgeons, and radiologists. The management of fistulizing Crohn’s remains an extremely challenging medical problem as many fistula do not respond to available treatments and new approaches are necessary.
Adult mesenchymal stem cells (MSCs), including adipose-derived mesenchymal stem cells (ASCs) are considered a promising tool for cell therapy in regenerative medicine or for treating inflammatory and autoimmune diseases due to their immunomodulatory capacity and paracrine effects through trophic factors with antifibrotic, anti-apoptotic, or pro-angiogenic properties [5, 6]. ASCs show immunomodulating properties and regulate the function of a broad variety of immune cells including B lymphocytes, T lymphocytes, NK cells, monocyte-derived dendritic cells, and neutrophils [7, 8, 9, 10, 11, 12, 13]. The specific molecular and cellular mechanisms rely on both cell contact-dependent mechanisms and paracrine effects through the release of various soluble factors including hepatocyte growth factor, prostaglandin-E2, transforming growth factor-β1, indoleamine 2,3-dioxygenase (IDO), nitric oxide, interleukin (IL)-10, IL-6, heme oxygenase-1, or human leukocyte antigen (HLA)-G5 . Among these factors, IDO activity appears to be a key player in human ASC-mediated immunomodulation .
ASCs have to be primed by inflammatory mediators, including interferon-gamma (IFNγ), IL1β, and tumor necrosis factor alpha (TNF) alpha which are strongly secreted by activated immune cells in inflamed environments (i.e., the perianal fistulas) in order to show their full immunomodulatory properties . In addition to the immunomodulatory properties, ASCs are considered immune privileged because: (1) they constitutively express only low levels of cell-surface HLA class I molecules and lack expression of HLA class II, CD40, CD80, and CD86; (2) after stimulation of ASCs by inflammatory mediators, there is upregulation of HLA class I and induced expression of HLA class II molecules on the ASCs, but without the expression of classic costimulatory molecules which would lead to anergy of T lymphocytes and immune tolerance; and (3) upon stimulation with inflammatory mediators released by activated immune cells, ASCs trigger an anti-inflammatory and immunomodulatory response (mainly through the induction of IDO) that may also modulate the immune recognition. These factors may contribute to a delayed or reduced activation of the innate and adaptive immune responses against allogeneic ASCs by the recipient’s immune system. This immune privilege of ASCs therefore supports the feasibility of allogeneic treatments without the requirement of suppression of host immunity [17, 18]. This can be beneficial as allogeneic ASCs obtained from a healthy donor provide a product that is accessible to more patients and avoids the need of collecting primary material in the patient. Thus, an “off-the-shelf” treatment can be rapidly administered from a completely validated cell bank.
The therapeutic benefit of human ASCs has been reported in a number of experimental models of inflammatory diseases including inflammatory bowel disease [19, 20]. In humans, local administration of autologous ASCs and bone-marrow mesenchymal stem cells has been used to treat perianal fistulas with promising results from a safety and efficacy perspective [21, 22, 23, 24]; however, no clinical use of allogeneic ASCs for this indication has been reported so far to our knowledge. Here, we present the results of the first phase I/IIa multicenter trial to assess the safety and efficacy of allogeneic expanded ASCs for the treatment of complex perianal CD fistulas.
Patients and methods
In this open-label, pilot study (ClinicalTrials.gov Identifier: NCT01372969), patients were selected according to the following inclusion criteria: age 18 years or older, diagnosis of CD at least 12 months before enrolment, presence of persistent and active complex perianal fistula with less than three fistulous tracts and/or external opening, non-active luminal CD defined by a Crohn’s Disease Activity Index (CDAI) ≤ 200, and good general state of health. The most relevant exclusion criteria were: patients with severe proctitis or dominant active luminal disease requiring immediate therapy; any abscess before start of treatment; presence of setons unless removed prior to start of treatment; rectal and/or anal stenosis; rectovaginal, anal, or non-perianal enterocutaneous fistula; treatment with infliximab or any other anti-TNF agent in the previous 8 weeks or tacrolimus or cyclosporine in the previous 4 weeks; any congenital or acquired immunodeficiency and allergy to anesthetics or magnetic resonance image scan (MRI) contrast.
From August 2009 to February 2010, a total of 34 patients were screened in six Spanish public hospitals. Of these, 24 patients were ultimately included in the study, as defined in the study protocol.
An MRI was performed at baseline to assess collections >2 cm associated with the fistula tract. Subjects initially received 20 million expanded adipose-derived allogeneic mesenchymal stem cells (eASCs) intralesionally in the fistula (see below). In the event of incomplete closure at 12 weeks, a further 40 million eASCs were administered. This dose proposal considers the no observed adverse effect levels established in the toxicological nonclinical studies from which we calculated the maximum safe starting dose in humans was calculated. Furthermore, we have positive safety and efficacy data with the same dose schedule from a series of clinical trials in fistulizing Crohn’s using autologous ASCs.
Subjects were followed until week 24 after the initial administration of cells. The primary endpoint was incidence of treatment emergent adverse events. Secondary endpoints were the reduction in the number of draining fistulas at 12 and 24 weeks; percentage of subjects in whom, at 12 weeks, the external openings of treated perianal fistula had closed; the increase in the number of closed fistulas at 12 and 24 weeks; percentage of subjects with MRI fistula healing at 12 and 24 weeks (absence of collections, >2 cm); changes over the time in the MRI Score of Severity (MSS); percentage of subjects presenting luminal relapse at weeks 12 and 24 and changes over the time in the Perianal Disease Activity Index (PDAI) and CDAI.
Fistula closure is defined as the absence of suppuration of the fistula through the external orifice, both spontaneously and upon application of pressure, with complete re-epithelization of the external orifice during clinical evaluation and absence of collections >2 cm, in three axis, directly related to the fistula tract treated, as measured by MRI.
Fistulas were evaluated at two consecutive visits, i.e., weeks 10, 12 and 22, 24 to avoid misinterpretation and guide treatment decisions. The treating physician evaluated fistula closure in all the corresponding visits. Nevertheless, in order to ensure unbiased fistula closure evaluation, a clinical evaluation was also performed by a second gastroenterologist/surgeon not involved in the direct care of the patient and with no access to the patient’s source records. Information on the fistula closure assessment was not exchanged between the evaluating physicians. Decisions related to performing a second cell administration the necessity for medical rescue therapy and/or the need to deviate from the prescribed standard treatment were made by the treating physician. Subjects were treated with standard of care during the entire study period, at the investigator’s discretion (excluding infliximab or other anti-TNFs, tacrolimus, or cyclosporine). Patient assessments included physical examination and vital signs with routine laboratory analyses, immunological assessment after treatment, rectosigmoidoscopy, MRI to identify the fistula tract anatomy, and any presence of complicating factors, CDAI and PDAI.
The Spanish Medicines Agency and the ethics committees of all participating centers approved the protocol and informed consent form before the inclusion of any patient in the study (EudraCT nr. 2008-007445-31). All patients gave written informed consent before participation. This study was conducted in accordance with the Declaration of Helsinki, in compliance with the consolidated Good Clinical Practice guideline set of the International Conference on Harmonization and the applicable regulatory requirements.
Isolation and expansion of eASCs
The allogeneic eASCs medicinal product consist of a cellular suspension of living adult stem cells of mesenchymal origin extracted from the subdermal adipose tissue of healthy donors. Subdermal adipose tissue was liposuctioned from the healthy donor and transported to the manufacturing facility. The donation, procurement, and testing were carried out according to the requirements of Directive 2004/23/EC and therefore under Directives 2006/17/EC and 2006/83/EC.
ASCs were isolated by digesting the adipose tissue with type I collagenase, followed by centrifugation. The cell pellet obtained was resuspended and lysed in erythrocyte lysis solution and centrifuged. The stromal vascular fraction, resulting from the cell pellet, was placed in cell culture containers in culture medium and antibiotics, and incubated at 37 °C and 5 % CO2 and in a humidified atmosphere. At 24–48 h post-plating, the culture medium was removed to eliminate the non-attached cell fraction. ASCs adhered to the plastic culture plates that were expanded under in vitro conditions. Every 3–4 days, the culture medium was changed after reaching 90–95 % confluence and the cells were detached with trypsin/EDTA, collected, centrifuged, and expanded without antibiotics to the required duplication. They were then harvested and cryopreserved until use.
Before the appointed administration date, sufficient cryopreserved vials were thawed to provide the required dose for administration. ASCs were recovered from their cryopreserved state by plating and culturing (to confirm viability). On the day when the vials were filled and packaged, the cultures were washed with phosphate buffer solution, and trypsin/EDTA. The ASCs were immediately resuspended in the selected excipients (Dulbecco modification Eagle medium and human albumin serum) to formulate the drug product.
The eASCs were characterized in terms of identity (phenotypic profile), purity, potency, morphology, viability, and cell growth kinetics according to the Guideline on Cell-Based Medicinal Products (EMEA/CHMP/410869/2006) and the Reflection Paper on Stem Cells (EMA/CAT/571134/2009).
The eASCs medicinal product was administered during major ambulatory surgery regimen according to the following procedure: (1) tract identification, with special emphasis on location of the internal opening; (2) tract curettage; (3) closure of internal opening with an absorbable suture; (4) cell resuspension used immediately to prevent cells from settling; and (5) administration of cell suspension with a fine, long needle into the tract walls, placing half of the total cell dose in the intersphincteric tracts and those adjacent to the internal opening, and the other half in the tract walls in the direction of the external opening. Injection was very superficial, no deeper than 2 mm.
Although sample size was fixed without test power considerations, the number of 24 evaluable patients was considered to be sufficient to fulfill the aim of the study. The primary outcome measure was the incidence of treatment-related adverse events (AE). This was assessed by calculating the proportion of subjects with at least one treatment-related AE, along with the corresponding binomial exact 95 % confidence intervals.
Efficacy variables (fistula closure at 12 and 24 weeks) were analyzed both in the full analysis (FA) and per protocol (PP) populations, the latter excluded patients with major protocol deviations. Quantitative variables were summarized by standard descriptive summary statistics (for absolute values and absolute changes from baseline). Likewise, the Wilcoxon signed-rank test was applied for each visit change in order to determine response changes. Frequency tables were calculated for categorical data. Likewise, baseline to postbaseline shift tables were also calculated to show the frequencies of patients shifting among the analyzed categories (Stuart–Maxwell test). The SAS System v8 was used for the statistical analysis (SAS Institute Inc., Cary, NC, USA).
Out of a total of 34 screened patients, 24 were ultimately included in the study and received the study treatment. Reasons for screening failures were noncompliance with inclusion/exclusion criteria (seven cases), withdrawal of informed consent (two cases), and investigator decision (one case).
The FA population or the intention-to-treat population (ITT) refers to all subjects who received at least one dose of study treatment while the PP population included all patients who met all inclusion criteria and none of the exclusion criteria, received at least one dose of study treatment, and completed the study without any major protocol deviation. The safety analysis was based on the ITT population and the efficacy analysis was done both on the ITT and PP.
Demographic and baseline characteristics
N = 24
11 (45.8 %)
Number of fistula tracksa
15 (62.5 %)
6 (25.0 %)
3 (12.5 %)
Number of external openingsa
18 (75.0 %)
4 (16.7 %)
2 (8.3 %)
1 (4.2 %)
5 (20.8 %)
1 (4.2 %)
17 (70.8 %)
19 (86.4 %)
3 (13.6 %)
Rectum wall involvementa
1 (4.2 %)
5 (20.8 %)
18 (75.0 %)
Presence of ulcersa
20 (83.3 %)
4 (16.7 %)
20 (83.3 %)
4 (16.7 %)
18 (75.0 %)
6 (25.0 %)
Presence of narrowingsa
21 (87.5 %)
2 (8.3 %)
1 (4.2 %)
Number of affected segmentsa
19 (79.2 %)
5 (20.8 %)
Of the 24 treated patients, 16 patients completed the study period and eight were prematurely withdrawn for various reasons. Two patients dropped out due to protocol deviations (one patient received antibiotic treatment for more than 4 weeks and another patient did not perform the screening pregnancy test at screening or visit 1). Other premature withdrawal reasons were presence of adverse event (perianal abscess) in two cases and outbreak of the underlying Crohn’s disease in four cases.
Treatment-emergent adverse events and related treatment-emergent adverse events during the study (safety population)
System organ class
n = 24
Treatment emergent adverse events (in more than one patient)
General disorders and administration site conditions
Infections and infestations
Anal fistula infection
C reactive protein increased
Musculoskeletal and connective tissue disorders
Related treatment emergent adverse events
Only two serious adverse events were reported in the study, “pyrexia” and “perianal abscess”. In the investigator’s opinion, these events were considered to be possibly related to the study treatment and both patients were withdrawn from the study. No clinically relevant abnormalities were found during the physical examination or in the vital signs.
Reduction from screening in the number of draining fistulas after 24 weeks
4 (33.3 %)
4 (30.8 %)
Yes (at least 1)
8 (66.6 %)
9 (69.2 %)
Reduction from screening in the number of draining fistulas after 12 weeks
7 (36.8 %)
8 (40.0 %)
Yes (at least 1)
12 (63.1 %)
12 (60.0 %)
Closure of external openings after 12 weeks
12 (63.2 %)
13 (61.9 %)
Yes (at least 1)
7 (36.8 %)
8 (38.1 %)
Closure of external openings after 24 weeks
7 (46.7 %)
7 (43.8 %)
Yes (at least 1)
8 (53.3 %)
9 (56.3 %)
Fistula closure at 24 weeks (absence of suppuration, complete re-epithelization, and MRI absence of collections >2 cm)
13 (72.2 %)
14 (70.0 %)
5 (27.8 %)
6 (30.0 %)
Subjects presenting luminal relapse after 12 weeks
22 (100.0 %)
24 (100.0 %)
0 (0.0 %)
0 (0.0 %)
Subjects presenting luminal relapse after 24 weeks
17 (77.3 %)
19 (79.2 %)
5 (23.7 %)
5 (20.8 %)
A total of seven patients (36.8 %) showed closure of the external opening of the treated perianal fistula in the per-protocol population and a total of 8 patients (38.1 %) in the full analysis population at week 12. A total of eight patients (53.3 %) showed closure of the external opening of the treated perianal fistula had closed in the per-protocol population and a total of nine patients (56.3 %) in the full analysis population at week 24 (Table 2).
The independent investigator’s assessment (using a combination of clinical and MRI assessment) confirmed a total of five (27.8 %) patients in the PP and six (30 %) in the FA showed closed fistula at week 12, and similar results were shown for fistula closure at week 24. This combined assessment was performed using the definition of fistula closure as “the absence of suppuration of the fistula through the external orifice, both spontaneously and under pressure, and complete re-epithelisation of the external orifice in the clinical evaluation and absence of collections >2 cm, in three axes, directly related to the fistula tract treated, as measured by MRI”.
Changes over time in the MSS
Wilcoxon P value
Wilcoxon P value
Symptomatic perianal fistula has a major negative impact on quality of life in patients suffering from Crohn’s disease and, unfortunately, treatment of complex perianal fistula remains unsolved. Although antibiotics are commonly used as first-line therapy , their efficacy has never been adequately demonstrated in randomized clinical trials (RCT) . Indeed, there is a general consensus that antibiotics are useful to control the septic foci but do not exert a prolonged effect. Immunosuppressants (IMMs) are commonly used as second-line therapy, but again no RCT assessing the efficacy of this treatment exists. Data has been derived from post hoc analyses of RCT in which IMMs were used for treatment of active luminal disease. A meta-analysis of results with IMMs showed efficacy in terms of achieving response (closure or reduction in drainage) but no data assessing the recommended endpoint of remission of fistulizing disease could be analyzed .
Recently, several anti-TNF monoclonal antibody treatments have been approved to treat patients with Crohn’s disease. To date, only the monoclonal antibody infliximab has demonstrated efficacy for the treatment of anal fistula in Crohn’s disease patients in a RCT . However, a large number of infliximab-treated patients continue to suffer from disease activity and high relapse rates , whereas only a small percentage of them have complete fistula healing [30, 31]. A major disadvantage of anti-TNF therapies is the risk for a variety of major adverse events such as serious infections, serious infusion reactions, hepatitis B virus reactivation, and congestive heart failure. In addition, neutralizing antibodies to the monoclonal antibodies can develop and limit or eliminate effectiveness of long-term use. To minimize the development of antibody formation, other immunomodulators like azathioprine, mercaptopurine, cyclosporine or methotrexate, and corticosteroids are frequently used concomitantly with the anti-TNF therapy increasing the risk of the patient of adverse events.
Surgery is an additional option. The most common surgical treatments include: incision and drainage (if an abscess is detected), placement of loose (draining) setons, occasionally local surgery repair (fistulotomy, flap repair), and, in extreme cases, proctectomy (removal of the colon and rectum) [32, 33, 34].
Conflicting data is available on the success rates of instillation of fibrin glue and anal fistula plugs and remained limited to small, uncontrolled studies and case series making it difficult to recommend the utility of these procedures in patients with Crohn’s disease and perianal fistula . One recent randomized controlled trial reported on the outcome of fibrin glue treatment in complex versus simple fistulas in patients with non-active inflammatory Crohn’s disease . In this study, clinical remission defined as the absence of draining fistula was much lower in the complex fistula group as compared to the simple fistula group (25 versus 50 %).
A large body of evidence demonstrated that ASCs have immunomodulatory and anti-inflammatory properties. The differentiation properties of ASCs are highly reduced during the expansion process while the immunomodulatory and anti-inflammatory effects appear to be intrinsic and unaltered by the expansion. It is well documented that the administration of eASC induces a reduction of the inflammatory pattern [5, 37]. Additionally, eASCs, are considered to be immune privileged, allowing the administration without HLA matching and without the need for immunosuppression [38, 39, 40]. Indeed, allogeneic eASCs exert a local anti-inflammatory and antiproliferative effect at the inflammation sites.
Adult stem cell therapy has promising application in the treatment of fistulizing Crohn’s disease with a combination of anti-inflammatory and regenerative effects as the proposed mechanism of action. To achieve the desired therapeutic effect, the cells need to be properly administered into the tracts together with tract curettage and the internal fistula openings need to be closed. Local administration of the cells along the fistula tract enables the full dose to exert its effect directly at the site of inflammation, without dilution. Local administration is preferred over systemic routes to adequately treat this condition. Indeed, Allesandroni et al.  recently discussed the local administration of infliximab as a possibility to treat patient not responding to intravenous administration.
Mesenchymal stem cells locally injected in the fistula have shown to be safe and effective in an autologous setting [21, 22, 23, 24]. The first mesenchymal stem cell administration published in the literature was a case report of a 33-year-old woman with CD and a rectovaginal fistula refractory to treatment. One week after administration of ASCs, the fistula had completely closed and no recurrence was reported in the 3 months of follow-up . A subsequent phase I trial was conducted in five patients with fistulas in Crohn’s disease. Nine eASCs administrations were performed in four patients with fistulizing Crohn’s disease. Complete closure of the external opening and no spontaneous or pressure suppuration was noted in 75 % of the cases. The remaining 25 % showed nevertheless a partial closure of the external opening, with a decreased discharge flow after a similar follow-up. No adverse effect related to the product was observed in any patient at the end of the follow-up period (at least 12 months) . In a subsequent phase II study, again with autologous ASCs, the proportion of patients whose fistulas were closed was significantly higher with autologous eASCs (p < 0.001). This efficacy was observed in both Crohn’s and non-Crohn’s subpopulations. No statistical difference between the ASC and control group was observed for the CD subpopulation. However, the lack of statistical significance could be due to the low number of CD patients. Nonrelated severe adverse events were found in the study .
Furthermore, Ciccocioppo et al. recently investigated the feasibility, safety, and efficacy of serial intrafistular injections of autologous bone marrow-derived mesenchymal stromal cells in 12 consecutive patients with fistulizing Crohn’s disease. Sustained complete closure (seven cases) or incomplete closure (three cases) of fistula tracts was observed with a parallel reduction of Crohn’s disease and perianal disease activity indexes (p < 0.01 for both), and induction of rectal mucosal healing without any adverse effects .
Allogeneic stem cells easily obtained from a healthy donor provides a product accessible to more patients and avoids the need of collecting primary material from patients. Thus, an easily available treatment can be rapidly administered from a completely validated cell bank and provides an economically affordable therapy to large numbers of candidate patients.
Allogeneic mesenchymal stem cells have now been used therapeutically and safely in a large number of clinical trials . By providing the required levels of safety and efficacy, allogeneic ASCs could provide an off-the-shelf cell therapy product, avoiding the inconveniences and costs associated with the production of an autologous therapy. This is the first trial of eASCs for the treatment of complex perianal fistula in perianal Crohn’s disease.
Although this phase II study was not powered to confirm efficacy objectives, and there was no control arm, the efficacy outcomes were nevertheless interesting. After 24 weeks follow-up, 69.2 % (FA) of patients had one or two tract reduction in the number of initially draining tracts, 56.3 % (FA) of eASC-treated tracts had their external opening closed with no difference between those patients who received one dose or two doses, and 30.0 % (FA) of patients had the fistulous tract closed. No patient showed collection(s) >2 cm in MRI at 12 or 24 weeks follow-up. Regarding MRI Score of Severity over time, for both populations, a reduction of the MSS during the whole study was reported (statistically significant at week 12 when compared to the screening value). Thus, clinical results seem to confirm that eASC-treated tracts experienced successful outcomes. On the other hand, these results show that nontreated tracts did not develop well in terms of the fistulous disease complete closure.
Our interpretation of the mechanism of action of eASCs is that they allow the fistula tract to heal due to (1) an anti-inflammatory effect, due to the production of immunomodulatory signals that reduce the chronic inflammation in the fistula by means of the activation of the eASCs by inflammatory mediators (mainly IFNγ) and production of IDO; and (2) paracrine effect, due to the release of trophic factors with regenerative properties (i.e., antifibrotic).
Patients receiving biologic agents were excluded to ensure that the safety findings were not influenced by possible adverse events, which are not infrequent with such therapies. The lack of control of the luminal disease was the reason behind the vast majority of the cases of loss to follow-up which indeed could have an impact on the efficacy analysis of the study.
The study also included some endpoints related more to the underlying luminal CD to evaluate to which extent a systemic effect of the cells could have occurred and could indeed be measured by means of CDAI. No major effect of the cells in the current dosage was found which could be linked to idea that the local effect of the cells is the primarily activity of the intrafistula injected cells.
Over the 24-week study, allogeneic eASCs demonstrated a good safety profile, with a low number of treatment-related adverse events (five patients) and a low number of serious adverse events (two patients). Most of the adverse events assessed by the investigators as related to the study treatment could be, by their nature, possibly more related to the study surgical procedure than to the study treatment itself (three anal abscesses and one case of pyrexia). The two serious adverse events (pyrexia and perianal abscess) may have been related to the cell administration procedure (tract preparation) and the patients subsequently completely recovered with no clinically relevant abnormalities during physical examination or in vital signs. The patient with perianal abscess that was withdrawn from the study nevertheless completed treatment.
Regarding the case of uterine leiomyoma, it should be taken into account that uterine fibroids are noncancerous growths of the uterus that often appear during childbearing years and are not associated with an increased risk of uterine cancer and almost never develop into cancer. In addition, the investigator decided to administer a second eASCs treatment despite his assessment that the adverse event was treatment related, indicating that a causal relationship was not clearly established. In summary, in view of these considerations, this study did not reveal any specific signs of tumorigenicity.
Some studies have reported transformation of in vitro expanded MSCs [42, 43]; however, these results were later retracted as they were shown to be related to cross-contamination with exogenous cancer cell lines [44, 45]. It is generally accepted that, if MSCs are expanded carefully and harvested at a population doubling far from senescence (as is the case of our ASC preparations), the risk of adult human MSCs becoming immortal and tumorigenic is very low compared to stem cells from other sources (embryonic or induced pluripotent). So far, clinical experience in many trials around the world does not indicate that tumorigenicity of MSC-based therapy represents a primary risk .
From the preclinical experience with this product (internal data TiGenix), no concern on potential tumorigenicity arose from the in vitro tumorigenicity studies as (1) no aberration could be detected in any sample based on the karyotype analysis; (2) no concerns arose from the C-myc expression, telomerase activity, and soft agar tests; and (3) the growth of all ASCs used in the preclinical program was monitored and PDs were established, showing a linear growth. The PD upper limit was established within the routine manufacturing process in order to avoid any potential senescence phase of the cells.
Furthermore, the extensive animal safety database generated as part of the GLP-controlled animal toxicity and tumorigenicity studies (internal data TiGenix) clearly demonstrate the lack of any tumor formation as no toxicity or specific adverse effects upon administration were found, including ectopic growth tissue, neoplasias, tumor development. The available database consists of different animal species (900 rats, 100 mice, and 38 rabbits) which have been inoculated with the expanded adipose-derived stem cells through various administration routes (subcutaneous, intravenous, and perianal administrations). Immunocompromised animals were used which are highly sensitive to tumors because of the compromised immune system. Follow-up ranged from 5 to 14 days to 3–6 months post administration, which is largely beyond the classical 14–28 days evaluation period.
The present novel therapy would provide significant benefits over current clinical management; the proposed tract treatment is a simple outpatient procedure and therefore, post-operative hospitalization is reduced. The proposed tract treatment is minimally invasive, ensuring significantly higher patient comfort. Furthermore, the ease of administration of the procedure should translate into better success rates, as opposed to surgery, where success is highly dependent on the surgeon’s skills. The proposed treatment will provide an easily accessible optimal treatment to all anal fistula patients. It has shown an acceptable safety profile with a low number of serious adverse events and could provide a first real closure alternative for anal fistulas due to the special tract preparation and the potent local anti-inflammatory effect of the eASCs administration. In addition, the proposed treatment exerts immunomodulatory effects over the abnormal lymphocytes, which provoke inflammation in the fistulas associated with Crohn’s disease. The cells can help locally control the deleterious impact of the underlying disease inside the fistula tract. This treatment could potentially eliminate the need of the maintenance course of infliximab or other systemic anti-TNF therapies, in cases of perianal CD with fistula because of the maintenance of the effect over time. Recurrence could potentially be decreased due to closure of the fistula tract and consequently the risk of incontinence associated with surgery will be eliminated (after one or two stages of anal fistulotomy, the risk of fecal incontinence ranges from 30 to 40 % [47, 48] and options that minimize sphincter injury are demanded ). Additionally, tumor incidence associated with recurrent fistula  could be minimized by reducing fistula recurrence.
In summary, these clinical results of eASCs show that eASCs are a safe treatment for fistulas that could overcome most of the problems associated with surgery and systemic anti-TNFs currently used for the management of perianal fistulas. The eASCs also demonstrated a promising good outcome in terms of tract closure of the treated fistula.
In the treatment algorithm of multidisciplinary management of fistulizing Crohn’s, this cell-based approach might well be utilized when failures occur to antibiotics, IMM, or anti-TNF and could be applied in conjunction with concomitant treatments in order to control the luminal disease.
Additional studies are necessary to confirm in a controlled design the efficacy profile of the eASCs and to establish the best strategy for their administration, including concomitant medication and dosage.
The authors thank José Luis Bravo and Lydia Dorrego (TiGenix) for their support and contribution during the study conduct.
There are no competing interests
This study was sponsored by TiGenix (formerly Cellerix).