Mariano García-Arranz

A Phase I Clinical Trial of the Treatment of Crohn’s Fistula by Adipose Mesenchymal Stem Cell Transplantation

PURPOSEThe effective management of fistulas in patients with Crohn’s disease presents an extremely challenging problem. Mesenchymal adult stem cells extracted from certain tissues, such as adipose tissue, can differentiate into various cell types. Therefore, we have tried to use such cells to stimulate healing of Crohn’s fistulas.METHODSWe designed a prospective Phase I clinical trial, involving five patients with Crohn’s disease, to test the feasibility and safety of autologous stem cells transplantation in the treatment of fistulas. We also studied the expression of various cell markers and the growth rates of the lipoaspirate-derived cells that were used for transplantation.RESULTSOne patient was excluded because of bacterial contamination of cultured cells. We inoculated nine fistulas in four patients with autologous adipose tissue-derived stem cells at Passage 3 or earlier. Eight inoculated fistulas were followed weekly for at least eight weeks. In six fistulas, the external opening was covered with epithelium at the end of Week 8, and, thus, these fistulas were considered healed (75 percent). In the other two fistulas, there was only incomplete closure of the external opening, with a decrease in output flow (not healed; 25 percent). No adverse effects were observed in any patient at the end of the follow-up period (minimum follow-up,12 months; maximum follow-up, 30 months; follow-up average, 22 months).CONCLUSIONSTo our knowledge, this is the first report of a clinical trial of cell therapy using autologous stem cells obtained from a lipoaspirate. Our results indicate that our protocol is feasible and safe for the treatment of fistulas in Crohn’s disease. The number of patients included and the uncontrolled nature of Phase I clinical trials do not allow demonstration of the effectiveness of the treatment. However, the results of the present study encourage to perform further studies in Phase II.

Expanded Adipose-derived Stem Cells for the Treatment of Complex Perianal Fistula: a Phase Ii Clinical Trial

PURPOSE: The feasibility and safety of stem cell-based therapy with expanded adipose-derived stem cells (ASCs) has been investigated in a phase I clinical trial. The present study was designed as a phase II multicenter, randomized controlled trial to further investigate the effectiveness and safety of ASCs in the treatment of complex perianal fistulas. METHODS: Patients with complex perianal fistulas (cryptoglandular origin, n = 35; associated with Crohns disease, n = 14) were randomly assigned to intralesional treatment with fibrin glue or fibrin glue plus 20 million ASCs. Fistula healing and quality of life (SF-12 questionnaire) were evaluated at eight weeks and one year. If healing was not seen at eight weeks, a second dose of fibrin glue or fibrin glue plus 40 million ASCs was administered. RESULTS: Fistula healing was observed in 17 (71 percent) of 24 patients who received ASCs in addition to fibrin glue compared with 4 (16 percent) of 25 patients who received fibrin glue alone (relative risk for healing, 4.43; confidence interval, 1.74-11.27); P < 0.001). The proportion of patients with healing was similar in Crohns and non-Crohns subgroups. ASCs were also more effective than fibrin glue alone in patients with a suprasphincteric fistulous tract (P = 0.001). Quality of life scores were higher in patients who received ASCs than in those who received fibrin glue alone. At one year follow-up, the recurrence rate in patients treated with ASCs was 17.6 percent. Both treatments were well tolerated. CONCLUSION: Administration of expanded ASCs (20 to 60 million cells) in combination with fibrin glue is an effective and safe treatment for complex perianal fistula and appears to achieve higher rates of healing than fibrin glue alone.

Autologous expanded adipose-derived 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.

Background: Autologous adipose-derived stem cells may represent a novel approach for the management of complex fistula-in-ano. After successful phase I and II clinical trials, a phase III trial was performed to investigate the safety and efficacy. Design: In this multicenter, randomized, single-blind, add-on clinical trial, 200 adult patients from 19 centers were randomly assigned to receive 20 million stem cells (group A, 64 patients), 20 million adipose-derived stem cells plus fibrin glue (group B, 60 patients), or fibrin glue (group C, 59 patients) after closure of the internal opening. Fistula healing was defined as reepithelization of the external opening and absence of collection >2 cm by MRI. If the fistula had not healed at 12 weeks, a second dose (40 million stem cells in groups A and B) was administered. Patients were evaluated at 24 to 26 weeks (primary end point) and at 1 year (long-term follow-up). Results: All results are according to the “blinded evaluator” assessment. After 24 to 26 weeks, the healing rate was 39.1%, 43.3%, 37.3% in groups A, B, and C (p = 0.79). At 1 year, the healing rates were 57.1%, 52.4%, and 37.3 % (p = 0.13). On analysis of the subpopulation treated at the technique’s pioneer center, healing rates were 54.55%, 83.33%, and 18.18%, at 24 to 26 weeks (p < 0.001). No SAEs were reported. Conclusions: In treatment of complex fistula-in-ano, a dose of 20 or 60 million adipose-derived stem cells alone or in combination with fibrin glue was considered a safe treatment, achieving healing rates of approximately 40% at 6 months and of more than 50% at 1-year follow-up. It was equivalent to fibrin glue alone. No statistically significant differences were found when the 3 groups where compared. Clinical trials registration: www.clinicaltrials.gov, identifier NCT00475410; Sponsor, Cellerix SA.

Treatment of enterocutaneous fistula in Crohn’s Disease with adipose-derived stem cells: a comparison of protocols with and without cell expansion

BackgroundExpanded adipose-derived stem cells (ASC) have been shown to be effective in treating Crohn’s patients with enterocutaneous fistulas. It is possible that unexpanded cells corresponding to the stromal vascular fraction (SVF) may also be effective.Materials and methodsA subpopulation of patients from a previous proof-of-concept phase I study with enterocutaneous fistulas received autologous expanded ASCs. The same selection criteria for inclusion were applied to patients who underwent SVF implantation to treat enterocutaneous fistulas. After tract curettage, cell suspensions (either SVF cells from lipoaspirate or expanded ASCs) were injected into the tract walls, and the fistulous tract was sealed with fibrin adhesive (with or without cells).ResultsIn the series that received ASCs, four fistulas could be evaluated, and cure was achieved in three out of four cases. In the series that received SVF cells, four fistulas were evaluated, with cure achieved in one out of four cases.ConclusionsAlthough a comparison of case series cannot be considered firm evidence, a therapeutic protocol that uses expansion prior to implantation does seem to be more effective than one that uses SVF cells directly from a lipoaspirate sample.

Expanded adipose-derived stem cells for the treatment of complex perianal fistula including Crohn's disease

Background: Complex perianal fistulising disease is a distressing condition. In patients without Crohns disease, surgery is the mainstay treatment but faecal incontinence and recurrence are high. Infliximab is used in Crohns patients but not all respond to therapy. Objective: After an evaluation of the current treatment options, we discuss studies of adipose-derived stem cell (ASC) therapy, a novel approach for treating complex perianal fistulas. Methods: ASCs are obtained from a liposuction procedure and a subsequent expansion process. They are administered according to a strict protocol which involves infusion of the cells into the target lesion along with fibrin glue. Results/conclusions: A Phase IIb study comparing ASC and fibrin glue therapy with fibrin glue therapy alone showed that ASCs were effective at inducing healing in complex perianal fistulas.

Adipose-derived stem cells in Crohn's rectovaginal fistula.

Therapeutic options for recto-vaginal fistula in the setting of Crohns disease are limited and many data are available in the literature. The manuscript describes the history of a patient who has been the pioneer of our Clinical Trials in treating this disease in fistulizing Crohns disease environment. We believe it is the first time that a patient with this disease has been treated by adipose-derived stem cells in allogeneic form. The conclusion of our study with Mary is that the use of mesenchymal stem cells derived from adipose tissue is secure, either in autologous or allogeneic form. Furthermore, we have proved that if we use multi-dose and multiple applications on a patient, it does not produce any adverse effect, which confirms us the safety of using these cells in patients at least in the fistulizing Crohns disease environment.

Low doses of bone morphogenetic protein 4 increase the survival of human adipose-derived stem cells maintaining their stemness and multipotency.

Mesenchymal stem cells (MSCs) have emerged as important tools for cell therapy; therefore, identification of factors capable of governing their ex vivo expansion become essential. In this study we demonstrate that human adipose-derived stem cells (ASCs) express all components of the bone morphogenetic protein (BMP)/BMP receptor signaling pathway and respond to BMP4 inducing upregulated expression of its specific target genes Id1-Id4. Moreover, ASCs grown in a medium reduced in serum produce endogenous BMP4 that could affect autocrinely ASC growth. On the contrary, dorsomorphin, an inhibitor of BMP signaling pathway, decreases cell numbers yielded from ASC cultures in correlation with increased apoptosis and decreased cycling cells. Therefore, BMP4 emerges as a possible factor for ex vivo expanding human ASCs. Our results demonstrate that, as other morphogens, BMP4 effects on human MSCs are dose dependent. High doses significantly increased apoptosis and drastically reduced cell proliferation, whereas low doses of BMP4 (0.01-0.1 ng/mL) significantly increase culture cell content, reduce the number of apoptotic cells, and increase that of cycling cells. Further, treatment of human ASCs with low doses of BMP4 does not modify expression of Nanog and Oct4, two transcription factors involved in self-renewal and pluripotency of stem cells or avoid their osteogenic or osteoblastic differentiation capacities when cultured in adequate inducing media, as shown by the induction of specific gene expression (CEBP, PPARγ, and RUNX2). Our results therefore support BMP4 as a promising factor for expanding human adipose tissue-derived MSCs maintaining their properties of stemness and multipotency.

Recurrent anal fistulae: Limited surgery supported by stem cells

AIM To study the results of stem-cell therapy under a Compassionate-use Program for patients with recurrent anal fistulae. METHODS Under controlled circumstances, and approved by European and Spanish laws, a Compassionate-use Program allows the use of stem-cell therapy for patients with very complex anal fistulae. Candidates had previously undergone multiple surgical interventions that had failed to resolve the fistulae, and presented symptomatic recurrence. The intervention consisted of limited surgery (with closure of the internal opening), followed by local implant of stem cells in the fistula-tract wall. Autologous expanded adipose-derived stem cells were the main cell type selected for implant. The first evaluation was performed on the 8(th) postoperative week; outcome was classified as response or partial response. Evaluation one year after the intervention confirmed if complete healing of the fistula was achieved. RESULTS Ten patients (8 male) with highly recurrent and complex fistulae were treated (mean age: 49 years, range: 28-76 years). Seven cases were non-Crohns fistulae, and three were Crohns-associated fistulae. Previous surgical attempts ranged from 3 to 12. Two patients presented with preoperative incontinence (Wexner scores of 12 and 13 points). After the intervention, six patients showed clinical response on the 8(th) postoperative week, with a complete cessation of suppuration from the fistula. Three patients presented a partial response, with an evident decrease in suppuration. A year later, six patients (60%) remained healed, with complete reepithelization of the external opening. Postoperative Wexner Scores were 0 in six cases. The two patients with previous incontinence improved their scores from 12 to 8 points and from 13 to 5 points. No adverse reactions or complications related to stem-cell therapy were reported during the study period. CONCLUSION Stem cells are safe and useful for treating anal fistulae. Healing can be achieved in severe cases, sparing fecal incontinence risk, and improving previous scoring.

Current and Emerging Applications of Droplet Digital PCR in Oncology

The clinical management of cancer has evolved in recent years towards more personalized strategies that require a comprehensive knowledge of the complex molecular features involved in tumor growth and evolution, and the development of drug resistance mechanisms leading to disease progression. Droplet digital PCR (ddPCR) has become one of the most accurate and reliable tools for the examination of genetic alterations in a wide variety of cancers because of its high sensitivity and specificity. ddPCR is currently being applied for absolute allele quantification, rare mutation detection, analysis of copy number variations, DNA methylation, and gene rearrangements in different kinds of clinical samples. This methodology has proven useful for the evaluation of archival tumor tissues, where poor DNA quality and limited sample availability are major obstacles for standard methods, providing less subjective and more automated quantitative results. However, most applications of ddPCR in cancer are focused on liquid biopsies (including cell-free DNA as well as circulating tumor cells) because these represent non-invasive alternatives to tissue biopsies that can more accurately reflect intratumoral heterogeneity and track the dynamic changes in tumor burden that occur in response to treatment at different times during follow-up. A broad spectrum of molecular markers have been interrogated in blood using ddPCR for diagnostic, predictive, and monitoring purposes in various malignancies. Emerging alternative approaches using other body fluids such as cerebrospinal fluid and urine are also currently being developed. This article aims to give a complete overview of ddPCR applications for molecular screening in oncology.

Treatment of Crohn's-Related Rectovaginal Fistula With Allogeneic Expanded-Adipose Derived Stem Cells: A Phase I–IIa Clinical Trial

The aim of this clinical trial was to determine the safety and feasibility of expanded allogeneic adipose‐derived stem cells to treat Crohn’s‐related rectovaginal fistula (CRRVF). We designed a phase I–II clinical trial (https://ClinicalTrials.gov, NCT00999115) to treat 10 patients with CRRVF. Patients receiving biological therapy during follow‐up were excluded. Curettage was performed, and a vaginal or rectal flap was added if the surgeon considered it necessary. The therapeutic protocol included intralesional injection of 20 million stem cells in the vaginal walls (submucosal area) and fistula tract. Healing was evaluated 12 weeks later. If the fistula had not healed, a second dose of 40 million stem cells was administered. Patient follow‐up was 52 weeks from last cell injection. Healing was defined as re‐epithelialization of both vaginal and rectal sides and absence of vaginal drainage. Cytokines and immunological blood tests were monitored. Serious adverse events or rejection issues were not observed. Five patients were excluded because biologic drugs were required to treat a Crohns disease flare‐up during follow‐up. Cytokine profiles and immunotoxicity assays showed no statistically significant alterations. Sixty percent of the nonexcluded patients achieved a complete healing. Expanded allogeneic adipose‐derived stem‐cell injection is a safe and feasible therapy for treating CRRVF, and the healing success rate seems promising (60%). The results of this trial encourage further exploration into this therapy.