Two phase I/II clinical trials for the treatment of urinary incontinence with autologous mesenchymal stem cells

Abstract We evaluated the safety and feasibility of adipose‐derived mesenchymal stem cells to treat endoscopically urinary incontinence after radical prostatectomy in men or female stress urinary. We designed two prospective, nonrandomized phase I‐IIa clinical trials of urinary incontinence involving 9 men (8 treated) and 10 women to test the feasibility and safety of autologous mesenchymal stem cells for this use. Cells were obtained from liposuction containing 150 to 200 g of fat performed on every patient. After 4 to 6 weeks and under sedation, endoscopic intraurethral injection of the cells was performed. On each visit (baseline, 1, 3, 6, and 12 months), clinical parameters were measured, and blood samples, urine culture, and uroflowmetry were performed. Every patient underwent an urethrocystoscopy and urodynamic studies on the first and last visit. Data from pad test, quality‐of‐life and incontinence questionnaires, and pads used per day were collected at every visit. Statistical analysis was done by Wilcoxon signed‐rank test. No adverse effects were observed. Three men (37.5%) and five women (50%) showed an objective improvement of >50% (P < .05) and a subjective improvement of 70% to 80% from baseline. In conclusion, intraurethral application of stem cells derived from adipose tissue is a safe and feasible procedure to treat urinary incontinence after radical prostatectomy or in female stress urinary incontinence. A statistically significant difference was obtained for pad‐test improvement in 3/8 men and 5/10 women. Our results encourage studies to confirm safety and to analyze efficacy.

of male patients develop incontinence, 1,2 and after radical prostatectomy, this figure ranges from 5% to 60%. 3 Prostate cancer is the most frequently diagnosed type of cancer in men. 4 Female stress urinary incontinence (SUI) is the involuntary leakage of urine during events that result in increased abdominal pressure in the absence of a bladder contraction due to effort or exertion or on sneezing or coughing. 5 In 2017, estimates of its prevalence in the female population range from 10% to 40%. 6,7 SUI is associated with significant impairment in quality of life and has a significant socioeconomic impact, with costs steadily increasing as the population ages. 8,9 After radical prostatectomy, urinary sphincter injury is thought to be the main cause of urinary incontinence, as supported by urodynamic studies. 10,11 Many therapeutic options in male and female urinary incontinence have been used for treatment, with varying degrees of success; these include pelvic floor rehabilitation, bulking agents, slings, and artificial urinary sphincters. [12][13][14] The cost of the disease borne by health systems is substantial. 15 New approaches are being considered to improve the treatment, but its development is still uncertain (eg, 3D bioprinted muscle 16 or tissue engineering 17 ). Mesenchymal stem cells (MSCs) have demonstrated, in experimental models, safety and efficacy against urinary incontinence. 18 But not only MSCs have demonstrated their efficiency in experimental models; in recent years, the proteins secreted by MSCs are generating interesting expectations for the treatment of urinary incontinence. 19,20 Numerous safety clinical trials have also been developed to analyze the safety of different MSCs for the treatment of urinary incontinence 21 ; unfortunately, there are many differences between cellular origin, application system, and follow-up.
The ability of adult MSCs from adipose tissue, called adiposederived stem cells (ASCs), to differentiate into several cell lines has been widely described. 22 Zuk et al demonstrated the capacity for myocyte differentiation in vitro when cultured next to myoblasts, 23 and an in vivo experiment in a model of ischemic muscular injury showed ASCs in 20% of myotubes in repair, thereby confirming that ASCs can participate in muscle repair if they are in a "myogenic environment." 24 Only two papers have been published on ASCs for urinary incontinence: one used stromal vascular fraction, and the other is a pilot study with ASC cultures combined with bovine collagen in female patients. 25,26 We present the results of two phase I-IIa trials to be registered at ClinicalTrials.gov in which autologous ASCs are used for urinary incontinence treatment after radical prostatectomy, and the first one for female SUI. In both, the main objective was to assess the feasibility of the process and the safety of the cell therapy, and the secondary objective was to obtain preliminary results about therapeutic efficacy. We designed two clinical trials in which a total of 10 patients per trial would be recruited. All patients presented urinary incontinence after radical prostatectomy in the men's trial and SUI in the women's trial, and after conservative treatment had failed ( Figure 1).

| MATERIALS AND METHODS
The mean age of the patients enrolled in the men's trial was 67.6 years, with an SD of 5.2; likewise, the incontinence evolution time was 60.5 months, with an SD of 24.5.
The mean age of the patients enrolled in the women's trial was 56.8 years, with an SD of 9.00; likewise, the incontinence evolution time was at least 12 months.
This study was designed to evaluate the feasibility and safety of ASC use and to produce preliminary efficiency results obtained using an inde-

Significance statement
This article reports the results of two clinical trials that studied safety of the treatment of urinary incontinence in men and women through the use of mesenchymal stem cells derived from adipose tissue obtained from the patient or cultured ex vivo.
skin and a small-drilled needle was inserted into the subcutaneous tissue. In all cases, we obtained 150 to 200 cc of adipose tissue.
The treatment began, in the men's clinical trial, with an initial implantation of 20 × 10 6 ASCs followed by a clinical evaluation after 3 months, and if significant improvement in incontinence was not achieved, a second implantation of 40 × 10 6 ASCs was performed. In the women's clinical trial, only a cell implantation of 40 × 10 6 ASCs was performed with the same follow-up.

| ASC preparation
The isolation of ASCs from lipoaspirate has been described previously. 27 We used MSCs derived from subcutaneous adipose tissue obtained by liposuction and processed by the Gregorio Marañón F I G U R E 1 Flowchart. A, The men's clinical trial, in which nine patients were recruited; before cell implant, one patient was excluded because of tumor diagnosis. No adverse events were observed. Significant improvement was achieved by three patients. B, The women's clinical trial, in which 10 patients were recruited; significant improvement was achieved by 5 patients. No adverse events were observed. ITT, intention-to-treat reasons, the doses of cells were cryopreserved in liquid N 2 (30% cell death: producer data). At least 1 week before the date of implantation, the cells were thawed and cultured. For administration, the cells were suspended in a sterile lactated Ringer's solution with 1% human albumin at 1 × 10 7 cells/mL. Samples were taken before release to examine viability, DNA stability, and pathogen controls (analysis performed by the producer). For implantation, 20 × 10 6 ASCs were prepared and 40 × 10 6 were cryopreserved for each male patient, in case they need a second dose, and 40 × 10 6 ASCs were prepared for each female patient.

| Treatment
In both trials, under direct vision and with the patient under sedation and in the lithotomy position, cell implantation was performed in the area close to the bladder neck and along the external sphincter using a compact cystoscope (17 Ch) and an endoscopic needle (7 Ch) for cell injection. A random distribution of cellular material was made, injecting volumes of 0.2 to 0.3 mL of ASCs at a depth of less than 0.5 mm; 7 to 8 injections (volume: 2 mL) were performed in all cases in the urinary sphincter of men and in women from the neck to the middle urethra.

| Follow-up
The feasibility of the process was assessed based on the absence of problems during sample collection, cell culture, and cell implantation, although in one case, the tissue from the liposuction was contaminated and liposuction had to be repeated because the fungal contamination could not be eliminated during cell culture.
Safety was assessed in terms of the incidence of adverse events and serious adverse events. Patients were monitored for adverse events at each study visit (implant, 4, 12, 24 weeks, and 1 year) and for any other observation that could alert of a possible abnormal/deleterious impact of ASCs. During follow-up, we analyzed changes in medical history, blood pressure, heart rate, temperature, systemic parameters (biochemical and hematological features), and local parameters (urine culture, uroflowmetry, cystoscope, and urodynamic evaluation). We also collected SF-36 and ICIQ-SF surveys.
Clinical response was defined as an objective improvement in urine leakage of more than 50% relative to baseline values as quantified by the pad test.

| Statistical analysis
The database was analyzed using the SPSS v.11.5 program (IBM, Armonk, New York). P values of <.05 were considered statistically significant. Values are shown in numbers and percentages. Quantitative values were compared by performing a temporal evolution study, and a before-after paired test was compared using a nonparametric Wilcoxon test (Wilcoxon signed-rank test).

| RESULTS
In the men's trial, 10 patients were recruited, one of whom was diagnosed with a pancreatic tumor after liposuction and was excluded Prostate cancer-diagnosed subjects via a biopsy and having had a radical surgery with a healing purpose in the previous 18 months.
Having urinary incontinence after the surgery.
Failure in any previous conservative treatment.

Inclusion criteria-women trial
Signed informed consent.
Good general state of health according to the findings of the clinical history and the physical examination.
Postmenopausal or over 18-years-old women taking highly effective contraceptives following the ICH (M3) EMA guide.
Women having rejected de-rehabilitation treatment or in whom the treatment had failed.
Genuine or combined stress urinary incontinence diagnosed with at least 1 year of evolution.

Exclusion criteria
Adjuvant therapy.
Prostate-specific antigen (PSA) ≥0.2 ng/dL after surgery. (Only men's trial) Present any sign or symptom that could indicate cancer progression. (Only men's trial) Present bladder outlet obstruction (means by uroflowmetry and urethrocystoscopy).
Active urine infection.
Alcohol or another addictive substances abuse during the 6 months before inclusion.
Presenting any other malignant neoplasia unless it is a basocellular or a skin epidermoide carcinoma or presents antecedents of malignant tumors, unless they are in a remission phase for the previous 5 years.
Cardiopulmonar illness, that by investigator decision, is instable or severe enough as to warrant patient exclusion.
Medical or psychiatric illness, that in the investigators opinion, could imply warrant patient exclusion.
Subjects with congenital or acquired immunodeficiencies, hepatitis B and/or C hepatitis, tuberculosis, or Treponema infection diagnosed at the moment of inclusion.
Pregnant or lactating women. (Only women's trial) Anesthetic allergy.
Major surgery or severe traumatism within over 6 months prior.
Administration of any drug under experimentation in the present or 3 months before recruitment. before cellular treatment. In another case, the cells were contaminated and liposuction had to be repeated, which left us with eight patients finally treated with cell therapy.
In the women's trial, 10 patients were recruited, all were treated, and all the follow-up was carried out without deviations from the protocol.
In  (Table 2). Case 2 went from 41 g of urine leakage to 9 g at final evaluation, and case 8 progressed from 287 to 123 g (Table 3). In the women's trial, all patients received only an injection of 40 × 10 6 ASCs ( Figure 1B). Cases 4-8 (5/10) showed an objective clinical improvement of more than 50% after 3 months of follow-up after 40 × 10 6 ASC treatment; this improvement, with no AEs, was confirmed by the absence of abnormalities in the uroflowmetry test and pad test performed during each visit and with an urethrocystoscopy at final evaluation ( Table 2).  (Table 3).

T A B L E 2 Urodynamics and voiding evaluation
In summary, in the men's and women's trials, three (37.5%) and five (50%) patients, respectively, showed an objective clinical improvement of more than 50%, constituting a statistically significant difference (P < .05). This improvement was maintained over time and showed in pad test and urodynamic evaluation.
We used the SF-36 and ICIQ-SF for the quality-of-life analysis and were unable to prove any statistical differences during follow-up in any trials.

| DISCUSSION
Surgical treatment is the gold-standard therapy for urinary incontinence, when noninvasive therapies have failed and short-term success has been achieved with injectable bulking agents. 28,29 The standard treatment for urinary incontinence in men is a conservative treatment during the first year after surgery, according to our experience and the recommendations of Kadoto et al, 30 and a second treatment with an artificial urinary sphincter. The standard treatment in women is tension-free suburethral mesh if the treatment by rehabilitation of the pelvic floor has failed. The success rate for those procedures in men is at least 80%, the complication rate is <10%, and the patient satisfaction rate is around 75% to 95%. The success rate for the procedure in women is more than 80% to 90% after more than 5 years of followup. However, these procedures have certain complications. 31 Complications, high cost, and not 100% long-term efficacy force us to seek alternative treatments for SUI. 32,33 Stem cell therapy has been investigated in different clinical applications. [34][35][36][37][38] Taking advantage of its capacity to induce tissue regeneration, stem cell treatment may be a promising strategy to overcome the current treatments for SUI. 39 In these studies, we used autologous cells to avoid the risk of rejection. Another important aspect of cellular therapy is the progressive normalization of the associated costs, thus making it an increasingly cost-effective option.
We believe that the low risk generated during outpatient liposuction followed by implantation of cells by endoscopic injection in the sphincter while the patient is under sedation, has advantages over traditional treatments. Moreover, the cost of both procedures (including liposuction and cell implant) is less than that of conventional treatments, mainly because the treatment occupies a short surgical room time and the patient does not need hospitalization. And this will be lower in the future if we can carry out allogenic treatments.
Sample collection, processing, and cell cultivation were performed under protocols designed by the research team and the production laboratory and were approved and validated by CEIC and AEMPS. It is noteworthy that cell expansion is performed in adherence of legislation and in licensed facilities, thus guaranteeing that the product administered to patients is homogeneous. In addition, unlike several previously described endoscopic treatments (bulking agents), we observed no cases of urethral structure and/or rigidity in the injection zones, nor did we find any alteration of voiding quality or worsening of incontinence as evidenced by uroflowmetry, urodynamic study, and pad tests performed on every visit. Furthermore, on the last visit, an urethrocystoscopy was performed on all patients to ensure the safety and effectiveness of intraurethral injections compared with the baseline.
Multiple parameters were collected for every patient before cell implantation and during every visit. We found no evidence of a systemic effect of cell therapy. More importantly, we observed no PSAlevel alterations in either the follow-up of any patient or in the subsequent routine follow-up (>3 years after cell therapy) in the men's trial.
We can thus state that cell therapy, when administered in selected patients who meet disease-free criteria, does not interfere with prostate cancer evolution, at least in our series of patients. Oncologic safety is a major issue in cell therapy, and several studies support this oncologic safety in time and support our clinical findings obtained over the short follow-up involved in this study. [40][41][42] The number of patients included does not allow us to make any statements on the effectiveness of the treatment. Probably the best way to evaluate urinary incontinence is the pad test and assessment of urine leakage through quality-of-life questionnaires. [43][44][45][46] We used two questionnaires and did not find any statistical differences with these tools, likely because they were not the best surveys and no patient was cured in the men's trial. However, we think that the SF-36 questionnaire is not the most appropriate to analyze the quality of life of patients with incontinence, and for future trials, we propose using the Incontinence Quality of Life Questionnaires (IQOL) Test or King's Health Questionnaire. 47 From an objective perspective, we used the number of pads per day, the 24-hour pad test, and urodynamic studies (Tables 2 and 3). During follow-up, we defined two time points for evaluation of therapeutic efficacy (3 and 12 months), making this decision in light of published accounts stating that, for endoscopic bulking agent treatments, initial response was lost after the disappearance of the bulking effect during the first 3 months. 14,28,48 The decision to schedule two cell doses was informed by the current lack of knowledge on optimal implanted cell dose. 49,50 Because of this and the fact that the maximum dose allowed by the AEMPS at the time of this trial was 40 × 10 6 ASCs, we decided on a dosage escalation program that called for 20 × 10 6 in the first injection and 40 × 10 6 in the second injection ( Figure 1) in the first clinical trial (men); owing to the excellent safety profile and to reduce costs, we decided to use only the highest dose in the second clinical trial (women). In addition, magnetic resonance imaging has been shown to be a good tool to evaluate the sphincter; it has not been considered in this phase I-IIa trial, but it could be useful in a phase II trial. There are many issues surrounding the mechanism by which stem cells act. Stem cells are known for their ability to differentiate into several cell lines, thus giving rise to their theoretical ability to restore damaged tissue. Furthermore, their paracrine effect has become more and more relevant over the last years; ASCs provide soluble mediators like cytokines and growth factors, which have effects upon cytoprotection, angiogenesis, tissue repair, and the normalization of the extracellular matrix and relief from inflammation. 52 If we can continue in this direction and answer all these questions, we will be poised to offer incontinence therapy that is safe, efficient, effective, minimally invasive, and lasting. According to this idea, in recent years, the secretome of ASCs for the treatment of urinary incontinence has been analyzed, and this way of working could inhibit some of the problems associated with the route of application, cell expansion/ dose, and so on; however, despite appearing promising, it is an almost unknown treatment. 53 Therefore, we can state that ASC therapy for urinary incontinence after radical prostatectomy or female SUI is safe and feasible, although its efficacy is relative, at least with the doses used in our trial and with a limited number of patients. We may conclude that 20 million ASCs are probably insufficient, although some patients had an obvious response; 40 million ASCs might be therapeutic for mild to moderate incontinence and have relative efficacy for moderate or severe incontinence. In any event, more trials to investigate the best cell dose are required.

| CONCLUSION
As our main conclusion, we can state that ASC therapy is a feasible and safe therapy from all points of view for the treatment of urinary incontinence in men and women, a finding that was the main objective of our trial. Both the direct repair effect and the paracrine repair effect of ASCs require further trials to achieve safety and efficacy for stem-cell therapy.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.