Ricerc@Sapienza

Urinary incontinence (UI) is extremely common (30-50% in female and 5-15% in male subjects) in all the world, able to significantly

impact on the quality of life of patients. It is also associated to a great distress influencing social life with high costs for both patients

and society (1,2). In particular, stress urinary incontinence (SUI) is related to urethral sphincter dysfunction . In male patients SUI is

mainly a complication of radical prostatectomy (RP) procedure for prostate cancer (PC)

Application of regenerative medicine using stem cells on the external urethral sphincter

Considering the poor results obtained using synthetic bulking agents to treat SUI, considerable interest inside regenerative medicine

has received the use of stem cells to functionally regenerate this structure in cases with SUI secondary to urethral sphincter

deficiency. Stem cell therapy (SCT) has been used to regenerate injured tissues and has been investigated in several diseases [3-5].

Differently to bulking agents, SCs could potentially repair the damaged sphincter. In a first step, SCs are isolated from autologous

human adult tissue biopsies and expanded in vitro. As second step, these cells are treated and prepared so to be injected some

weeks later to repair the musculature component of the urethral sphincter[3-5] .

How to choose suitable stem cells

Due to ethical consideration, immunological and oncological possible complications related to embryonal and umbilical cord stem

cells, research in SUI has mainly focused on adult stem cells. Different types of adult stem cells have been tested including

mesenchymal bone marrow stem cells (MSC), muscle-derive stem cells (MDSC) and adipose-tissue-derived stem cells (ADSC) [3-5].

MDSCs are easier and less painful to obtain in large quantities through a small skeletal muscle biopsy. Different studies showed

superiority of MDSC on MSC in terms of survival, engraftment, and improved tissue repair. MDSC have true stem cells properties,

excellent multipotent differentiation into myogenic, endothelial, neural lines. As autologous cells they do not cause an immunogenic

or allergic reaction and can persist longer than synthetic agents [3-5]. Another relevant advantage is their ability to become

innervated into the host muscle. In order to increase cell engraftment and survival, SCs can be transplanted with biomaterials such

as decellularized extracellular matrix (dECM) hydrogels, already tested in several clinical and preclinical studies.

Preclinical studies

Several preclinical studies, mainly on rat models, have been performed to support these experimental designs [6-9]. Almost all these

studies showed a regenerative effect of SCs when injected in the sphincter with differentiation in both smooth and striated muscle

cells. Electromyographic studies suggested that only MDSC are able to differentiate in functionally active myotubes and myofibrils

inside the sphincter tissue [6]. Chermansky et al [8] demonstrated a good incorporation of MDSC at 4 weeks into the striated muscle

of a damaged rat urethra. The SCs well differentiated in mature striate muscle, repairing the defect and increasing nervous support

in the sphincter. At 2-,4- and 6-week interval the leak point pressure (LPP) significantly increased in the treated rats when compared

to controls treated with saline solution.

Clinical studies

12 clinical trials [10-25] have been published on human subjects with SUI and MDSC were used in 9 studies. After in vitro culture

preparation, the amount of SC injected ranged from 1X 106 to 5.6X 107. Multiple circumferential Injections of SCs were mainly

performed through a transurethral endoscopic approach. Homogeneously all 12 trials did not describe severe side effects related to

the procedure and only 1 study reported on 10% of cases with obstructive urinary retention requiring temporary catheterization. A

meta-analysis using a random method described a whole event rate of continence recovery of 0.41 (95%CI 0.29 - 0.54) (I2 84.69% ;

Ministero dell'Università e della Ricerca

MUR - BANDO 2022

Q 107.50 - p<0.01) (Test of group differences p=0.96) with a high level of heterogeneity among studies.

Conclusions

Considering the relevant impact of UI influencing social life with high costs for both patients and society and the modest results

obtained using synthetic agents (collagen) to treat SUI, regenerative medicine approaches and the use of stem cells to functionally

regenerate the urethral sphincter should receive a higher level of interest [26]. The ambitious goal is to obtain a good integration of

the bioengineered cells into the tissue so to restore a functional organ.

A two-step safe procedure to obtain, prepare and inject autologous stem cells has been defined experimentally and MDSC or ADSC

showed the best characteristics to treat SUI. No experience in Italy have been developed and at now the procedure is only an

exciting experimental hypothesis.

2. Detailed description of the project: methodologies, objectives and results that the project aims to

achieve and its interest for the advancement of knowledge, as well as methods of dissemination of the

results achieved

This project purposes a prospective preclinical trial, preliminary to a following clinical trial, multicenter and multidisciplinary,

involving units and researchers at high level of clinical and experimental expertise on the topics involved in the project. In particular:

a. Urologic section with excellence and long term experience in the management of urinary incontinence in particular related to

prostate cancer treatments; b. Research and bimolecular section with long-term and international experience, for the management

and preparation in vitro of muscular derived and adipose derived stem cells; c. cellular biotechnology section with long term

experience to develop an animal (mouse) model for urinary incontinence and injection of stem cells and biomaterials in the

periurethral tissue. d. pathology section with excellence and long term and international experience in urogenital system,

immunohistochemical studies, tracking of biomaterials;e. imaging section with excellence and long term experience in magnetic

resonance studies, imaged tracking of implanted cells in animals.

Innovation points

The project purposes to develop a clinically applicable new therapeutic approach for the treatment of urinary incontinence (UI)

related to urethral sphincter deficiency in humans and in particular in male patients with SUI after radical prostatectomy for prostate

cancer, a disease at high level of social impact. The innovation is to use the regenerative medicine instead of synthetic materials,

collagen or artificial sphincters, through the injection of auto-transplanted muscle-derived stem cells (MDSC) or adipose derived

stem cells (ADSC) into the sphincter in combination with a pro-regenerative, clinically translatable, decellularized bioscaffold. The

ambitious goal is to obtain a good integration of the bioengineered cells into the tissue so to restore a functional organ. WE will

develop a useful and effective procedure to isolate and culture human MDSC and ADSC in vitro and we will preclinically verify the

procedure on mice, analyzing feasibility, engraftment, migration ,differentiation and tracking of cells inside the urethral sphincter.

These preclinical phases will be necessary to develop a following clinical trial on humans.

Study protocol

Our protocol will develop in two phases:

1. In vitro isolation, preparation and culture, of human MDSC and ADSC.

The protocol will be multicenter and multiunit, with a prospective enrolment of 15 human patients submitted to radical

prostatectomy (RP) for prostate cancer (PC).

Population

We will obtain approval from our internal ethic committee and all patients will be enrolled after informed consensus for all

procedures.

15 human patients with PC considered for surgery (RP) will be prospectively enrolled after informed consensus for the procedures.

Inclusion criteria: human patients aged 40 to 70 years old; male patients with histological diagnosis of non-metastatic prostatic

adenocarcinoma selected for radical prostatectomy as primary treatment following guidelines of the European Urologic Association

(EAU). The surgical procedure will be part of the common clinical practice and indication for the diagnosis of PC. The choice of this

type of patients to obtain muscle and adipose samples for our preclinical analysis is related to the fact that, in the future clinical trial,

these patients, in case they will develop post- surgery stress urinary incontinence (SUI), will represent the population for initial

biopsy and secondary autologous injection of MDSC or ADSC as regenerative treatment on the external urethral sphincter.

Exclusion criteria: neoplastic progression, previous or concomitant radiation, chemotherapy or other medical treatments that could

influence muscle activity. Neurological diseases or treatments that could influence muscle activity. Metabolic syndromes or disorders

Sample of MDSC and ADSC

In the 15 patients, at the beginning of the programmed surgery for primary treatment of PC disease, under anesthesia, a muscle

biopsy (0.5 cm3, 1-2 g of muscle fibers) and an adipose biopsy (0.5 cm3, 1-2 g of adipose tissue) from the abdominal initial incision

(muscle layer and subcutaneous fat tissue) normally used to access to the surgical field will be taken, and immediately transported

to the authorized center to prepare MDSC and ADSC. This technique will consent to safely obtain samples without further impact for

patients submitted to surgery as indication in our clinical practice.

Tissue sample and in vitro preparation

Muscle tissue will be isolated from connective tissue, minced into small pieces and enzimatically digested using two different

Ministero dell'Università e della Ricerca

MUR - BANDO 2022

solutions of collagenase: the first will be a Collagenase/Dispase solution (0.4 mg/ml in PBS) in a volume of 10 ml/g of muscle; the

second will be a Collagenase type II solution (1 mg/ml in PBS Calcium-Magnesium free), in the same volume. Both digestions will be

performed at 37 °C for 30 min in a water bath with agitation; finally digested muscle will be passed through a 70-μm cell strainer,

followed by 40-μm one.

Isolated cells will be centrifuged at 1200 rpm for 10 min, resuspended in RPMI 10% serum and plated at 2 × 10^6 cells/100 mm

plastic dish at 37 °C for 1 h with 5% CO2; non-adherent cells will be collected, centrifuged and plated again on plastic plates for 1 h

at 37 °C.

After the second pre-plating, non-adherent cells will be further collected, centrifuged and finally resuspended in RPMI 20% serum.

Then muscle cells will be plated into 100 mm gelatin or collagen coated gelatin dishes, at 10^6 cells/dish. Quality of cells will be

estimated using immunohistochemistry and immunofluorescence: in particular antibodies against desmin will be used to identify

myoblasts.

In general cellular production process will include: isolation, amplification, freezing and storage. Cells will be passaged before they

will reach 80% confluence and cultured both in autologous or fetal bovine serum as control.

The adipose tissue will be minced into small pieces and digested with 0.05%Trypsin/0.02% EDTA and 1 mg/ml collagenase type-I and

incubated at 37 °C at shaking water bath for 45 min. Tissue digestion will be blocked with PBS/20 % FBS and the tissue will be

filtered with a 100-μm cell strainer and the resulting cell suspension will be centrifuged twice at 377 g for 5 min and plated in

complete growth medium. Cells will be cultured in either regular standard condition (Low glucose-DMEM/1 %

penicillin–streptomycin/1 % glutamine/1 % nonessential aminoacids / 10 % FBS )or in serum-free GMP grade StemPro™ MSC cell

culture media (Giibco™), seeded in a T25 ventilated flask and incubated at 37 °C in 5 % CO2. To allow the initial adhesion to the

plastic surface of the flask, cells were left undisturbed in the incubator for 72 h. After 72 h the medium will be changed and cells will

be passaged when 70–80 % confluent. Quality control between the regular standard and clinically-translatable culture condition will

be performed at passage 3 and will include FACS analysis, colony forming unit fibroblasts assay (CFU-F) and trans-differentiation

assay. FACS analysis will be performed for mesenchymal (CD105, CD44, CD117, CD90), hematopoietic (CD45, CD117) and

endothelial (CD31) markers. CFU-F assay will be carried out plating hADSCs at low density (20–40 cells/cm2) in a 100 mm petri Dish

in both culture media. After 2 weeks of incubation, cells will be washed in PBS, fixed with 4 % paraformaldehyde and Giemsa-stained

and observed by optical microscope. Clusters of cells with a diameter ≥5 mm will be considered colonies. For trans-differentiation

assay ADSCs will be incubated with defined media for the three mesodermal lineage differentiation. ADCs will be plated in 6-well

plates at density of 5x 103, 1x 104 cells/cm2 cells and cultured with osteogenic (StemProOsteogenic Differentiation kit Gibco) and

adipogenic (StemPro Adipogenesis Differentiation kit, Gibco) medium, respectively. After 14 days of incubation, cells will be fixed

with 4 % paraformaldehyde. Calcium deposition will be analyzed by incubating the cells for 1 h at RT with a 2 % Alizarin red solution

at pH 4.1–4.3. Accumulation of lipid droplets will be evaluated by Oil Red Oil solution. For chondrogenic differentiation, 2.5x 105cells

will be resuspended in StemPro Chondrogenic Differentiation kit (Gibco) in a 15 ml-tube incubated in an upright position with media

that will be gradually added every 3 days avoiding pellet resuspension. After 3 weeks cells will be fixed with 4 % paraformaldehyde

and chondrospheres stained with 1 % Alcian blue 8GX solution to detect the presence of glycosaminoglycans.

…………..

2. Preparation and Injection of MDSC and ADSC into urethral sphincter of mice: analysis on survival, migration and differentiation

All experiments will be performed on 50 CD1 male wild type mice, 6 weeks old following EU and national regulations for animal

experiments (European Community Council Directive 86/609/EEC Nov 24,1986; Italian Ministry of Health on animal welfare law

116/92 on Care and Protection of living animals undergoing experimental procedures) and they will be approved by our Ethic

Committee. All efforts will be made in order to minimize the number of animals used and their suffering. In vitro experiments will be

carried out using GFP-labeled mouse-derived ADSC or MDSC as previously described. As bioscaffold, we will used a skeletal muscle

derived hydrogel which has been shown to increase cell engraftment, exert a pro-regenerative effect by increasing stem cell

differentiation with also modulating the inflammatory response of the host environment towards a pro-regenerative phenotype. This

bioscaffold is already available in Sapienza unit and recently used in clinical trials for myocardial regeneration with positive results.

Mice will be divided into 5 groups of treatment, which will be evaluated at 1-month ( 5 mice with MDSC alone, 5 mice with ADSC

alone, 5 mice with MDSC + scaffold, 5 mice with ADSC + scaffold and 5 mice with saline) and 3-month ( 5 mice with MDSC alone, 5

mice with ADSC alone, 5 mice with MDSC + scaffold, 5 mice with ADSC + scaffold and 5 mice with saline) after SC injection. One

month prior to surgery, adipose tissue will be obtained from the subcutaneous fat of the nap area under anesthesia and in vitro

prepared to obtain ADSC as previously described (see section 1. In vitro preparation). At the same time, an incision will be performed

on the gluteal area to reveal the gluteus muscle and a muscle biopsy will be performed. The tissue will be in vitro prepared as

previously described (see section 1 in vitro preparation) to obtain MDSC. Cells will be cultured for 3 weeks and sub-cultured 3 to 8

times. They will be subsequently counted, and the viabilities will be determined. Either for MDSC or ADSC an amount of

approximately 2.2-2.4X10^6 cells in either scaffold or saline solution to obtain a concentration of 50000 cells/μL will be prepared to

be injected in each mice.

The day of surgery (Time 0) the mice will be anesthetized and surgically the scrotum will be incised on the midline and the testicles

carefully displaced laterally. After dissection of surrounding tissue and fascia, the bulbar urethra will be identified, and the perineal

muscles visualized. A catheter will be inserted through the urethra to indicate the injection site. Injection of autologous prepared

cells (ADSC in 15 mice and MDSC in the other 15 mice) in their solution will be performed using a Hamilton syringe 27.5 G into the

periurethral tissue at the level of the mid urethra at 5 mm from the bladder neck divided in injection of 2 μL ( 50000 cells/μL each) at

the 3,6,9 and 12 o’clock position. After injection, surgical wounds will be sutured, and all mice will return to the cages.

At 1 and 3 months intervals, animals will be sacrified and bulbar urethra with the surrounding perineal muscles will be collected from

each mouse, fixed and samples will be sectioned on the cryostat in 18μM-thick serial sections, stained in hematoxiline eosine to

Ministero dell'Università e della Ricerca

MUR - BANDO 2022

recognize injection sites along with anatomic references such as urethra, external sphincter, perineal muscle, while potential fibrotic

area will be evaluated by Masson's trichrome staining. Immunofluorescence and histochemistry studies will also be performed to

further evaluated cellular phenotype. Sections will be rinsed in PBS, permeabilized with PBS containing 0.3% Triton X-100 and

washed three times in PBS. Non specific binding sites will be blocked by incubating sections 45 minutes at room temperature in 10%

normal goat or donkey serum in 0.3% PBS-Triton X-100. Slices will be immunoreacted overnight at +4°C in monoclonal mouse

anti-human desmin or rabbit plyclonal anti-Ki67. Desmin immunoreactivity will be considered as an early marker of muscle

differentiation. Alfa-bungarotoxin histochemistry will be performed to detect acetylcholine receptors in muscle

sections.Vascularization will be evaluated by CD31 and vWf expression while smooth muscle cells by smooth muscle actin

expression.

Semi-quantitative analysis to evaluate SC survival and density within the site of injection will be performed at 10-20x magnifications

under epifluorescence microscope and images will be processed. The site of injection will be outlined at the microscope thus to

obtain a measure of area and perimeter. Comparing groups at 1-mont and 3-month we will evaluate differences in quantitative

analysis and estimation of cell scattering along the muscular tissue, expressed as the largest value measured through the sections

for each sample for comparison.

In vivo tracking. To evaluate the best cell transplantation strategy as well as cell survival, both ADSCs and MDSCs will be transduced

with a lenti-viral construct, containing pLV-CMV-luciferin-GFP and transplanted in vivo either in scaffold or saline. In mice selected for

a long term evaluation (1-month and 3-month), at day 3, 7, 14, and 28 after transplantation, cells will be imaged used to monitor

printed CMPCs survival upon in vivo transplantation. Mice will be treated with 125 mg/kg of D-luciferin sodium salt (Promega) in PBS

via intra-peritoneal injection and imaged using IVIS-100 xenogen to detect transplanted cells.

Statistical analysis

STATA 1.7 software will be used and statistical significance will be fixed at less than 0.05. For the comparison of quantitative data

and pairwise intergroup comparisons of variables a Mann Whitney test will be performed. For comparison of qualitative data Fisher’s

Exact test and chi-square test will be used. Overall comparisons between groups were performed using a one-way analysis of

variance for within time-point analyses, and two-way analysis of variance

for across time-point analyses. Individual comparisons were made using Student’s t-test.

Results aimed to achieve

As primary, we aim to develop a clinically applicable procedure for sampling and for cellular isolation and expansion of autologous

MDSC and ADSC from male patients submitted to RP including: extraction, amplification, freezing, storage and suspension. We aim

to produce a significant amount of vital ADSC and MDSC higher than 2.5X10^7 and a useful suspension to be injected into the

urethral sphincter. We also aim to compare procedure results between ADSC and MDSC.

In the second phase on mouse model , we aim to demonstrate a valid periurethral injection of ADSC or MDSC in the animals, to track

cells position or migration from the site of injection, to demonstrate differentiation, viability and regenerative activity of the

transplanted cells and overall a functional improvement of SUI. Moreover, we want to evaluated for the first time, the use of a

clinically applicable skeletal muscle.derived hydrogel in an animal model and its effects on regeneration, cell engraftment and

differentiation..

All these data will consent to proceed in a following clinical trial on human male patients with SUI after RP.

Interest for advancement in knowledge

Our results will significantly improve knowledge related to: efficacy and safety of regenerative medicine with MDSC or ADSC to treat

SUI, ability to functionally regenerate the urethral sphincter using MDSC or ADSC injection, preparation and tracking of MDSC,

monitoring the method for a standardized injection, tracking of SC once injected so to integrate and regenerate the sphincter, safety

of auto-transplantation of MDSC and ADSC into the urethral sphincter, comparative analysis of efficacy in terms of preparation and

functional recovery in SUI between ADSC and MDSC

Methods for dissemination of results

Results will be published on a peer-reviewed journal at the highest IF level (Lancet Journal). Moreover, results will be disseminated

through presentations at the major national and international congresses related to urologic, stem cells, chemistry societies.

3. Project development, with identification of the role of each research unit, with regards to related

modalities of integration and collaboration

The protocol will be submitted for approval to our ethic committee. All multidisciplinary sections in the two units involved in the

project have been already identified and all have a high level of experience to conduct these experiments and to perform the

project. The section are: urologic section, biomolecular research section, animal model experiment section, chemical section,

pathology section, imaging section

Project development

- Identification of the male population and selection on the basis of inclusion and exclusion criteria: our urologic sections perform

approximately 50 radical prostatectomy per month. We consider obtaining the 15 samples from male patients in a 4-month period

(Unit 1,2)

- Muscle Sample, adipocyte sample, in vitro preparation: our biomolecular research sections, will receive biopsy samples regularly at

patient inclusion from month 1 and with the last patient estimated at month 4. Culture preparation of SC suspension will be obtained

Ministero dell'Università e della Ricerca

MUR - BANDO 2022

in 6-8 weeks and concluded for the last case into month 10. (Unit 1-2)

- Experiments on mice form the animal model experiment sections: biopsy sample for MDSC and ADSC will be obtained one month

before surgery and suddenly prepared to obtain adequate MDSC or ADSC solution for injection. Injection of the solution in the 50

mice will be performed one month after cells extraction. Monitoring on tracking, in vivo tracking using xenogen and magnetic

resonance imaging,differentiation, migration, regenerative activity, will be performed into 12 months. (Unit 1,2). Pathologic

examination on mice with immunofluorescence, histochemistry,semiquantitative evaluations with epifluorescence studies will be

performed at different intervals till 3 months.

- Elaboration of data: all data obtained from the two phases will be elaborate and controlled. Statistical analysis will be elaborated

and draft for publication will be complete. These procedures will be completed in the last 2 months (Unit1,2)

Role of each unit and all research sections involved

The two Units guarantee the presence of: urologic section, biomolecular research section, animal model experiment section,

pathology section, chemical section, imaging section

- Urologic sections (Unit 1,2): enrolment of the human population with SUI after RP. Definition of inclusion and exclusion criteria.

Muscle biopsy to obtain adequate samples for MDSC culture. Adipose biopsy to obtain adequate samples for ADSC. On mice

experiments, identification of the perineal muscle zone and injection of the SC solution into the periurethral tissue; catheterization of

the mice urethra. Recruitment of patients, evaluation of inclusion criteria and procedures will be conducted in collaboration between

Unit 1 ( Prof Alessandro Sciarra and Prof Stefano Salciccia in the department of Urology University Sapienza of Rome) and Unit 2

(Prof Enrico Finazzi Agro' and Prof Salvatore Sansalone, Department of Urology, University Tor Vergata of Rome) with the assistant of

other collaborations and consultants (Dr Gianna Mariotti). One research grant is asked to obtain a new person with high competence

involved in both in vitro and on animal experiments

- Biomolecular research sections, chemical section (Unit 1,2): in vitro experiments to produce MDSC and ADSC including: extraction,

amplification, freezing, storage and suspension. Muscle tissue isolation from connective tissue, myoblasts and adipocyte cells

isolation and preparation in culture. Incubation of cells and quality assessment. Use of antibodies to identify myoblasts and

adipocytes. Extraction and preparation of 2.5X10^7 cells for each injection. Definition and preparation of the tracking system to be

associated with MDSC and ADSC. Preparation of decellularized skeletal muscle scaffolds for in vivo transplantation with MDSC and

ADSC so to minimize inflammatory reaction at injection and to improve gradual delivery into the sphincter. In vitro experiments and

their evaluation will be centrally developed in the Department of Molecular Medicine, University Sapienza, by Dr Roberto Gaetani in

collaboration with other consultants Dr Elisa Messina with high experience on biomaterials and cells regenerative studies on muscle

tissue. One research grant is asked to obtain a new person with high competence involved on in vitro experiments

- Animal model experiment section, imaging section (Unit 1). Preparation of all experiments on mice. Extraction of muscle and

adipose cells from mice biopsy. Culture of muscle and adipose tissue to obtain MDSC and ADSC. Preparation of solution to be

injected in mice. Definition and performance of studies to follow MDSC and ADSC after injection and during follow-up.Tracking of

cells in mice. Xenogen and imaging studies and analysis to track injected cells into animals, evaluating, migration, implantation. All

experiments on animals will be conducted centrally in the SAIMLAL Department, Unit of histology and Embriology, Section on Animal

Models, University Sapienza of Rome in close collaboration with the team of Prof Antonio Musaro by Dr Laura Barberi and Dr Roberto

Gaetani with high experience on animal studies on muscle tissue. Workflow will include isolation of muscle and adipose cells from

mice biopsy, cell proliferation and quality assessment, injection into animal models and in vivo functional evaluation by imaging

analysis performed by Dr Gabriele De Luca Department of Molecular Biology Istituto Superiore Sanita' Rome. In vivo tracking

analysis will be performed by Xenogen to evaluate cell engraftment, survival and localization. Imaging tracking studies will be

conducted in the same department by Prof Valeria Panebianco from the department of Radiologic Sciences University Sapienza, with

high level experience in studies on magnetic resonance and urogenital system.

- Pathology section (Unit 1): preparation of section from mice models. Fixation and section of tissue from mice at cryostat.

Immunofluorescence and histochemical analysis on perineal muscle of mice. Epifluorescence analysis. Semiquantitative evaluation

on tissue preparations. All pathologic evaluations will be conducted centrally in the department of Radiologic and Pathologic Science,

University Sapienza, Rome, Prof Fabio Magliocca with high level experience on pathologic evaluation, histochemical and

immunofluorescence studies on urogenital system

Modalities of integration and collaboration

All units involved in the project are already used to collaborate together in trials and experimental projects and the two units

guarantee a complete composition of the group with urologic section, biomolecular research section, animal model experimental

section, chemical section, imaging section and pathology section. There is an already proved and functional connection among all

units. In particular regarding the present project, researchers from units and sections are well informed on all procedures, aims and

goals involved in the project. Collaboration among the clinical expertise of the urologic section to define population and to clinically

follow results ,with the experimental expertise on SC preparation and quality control of the biomolecular research section, animal

model expertise to prepare all experiments on mice from the animal model, xenogeny imaged analysis and imaging experiments to

check target injection and morphologic modifications of the sphincter during follow-up and the tracking expertise to follow

integration and regenerative properties of MDSC into the muscle sphincter will guarantee high quality and multidisciplinary

integrated results.

Ministero dell'Università e della Ricerca

MUR - BANDO 2022

Results obtained will be evaluated and prepared through an integrate and multidisciplinary collaboration among all units so to cover

all aspects involved in the project ranging from the research application to the development of a clinically applicable new

therapeutic product.

4. Possible application potentialities and scientific and/or technological and/or social and/or economic

impact

Urinary incontinence (UI) is extremely common in all the world, able to significantly impact on the quality of life of patients. It is also

associated to a great distress influencing social life with high costs for both patients and society. Considering the poor results

obtained using synthetic bulking agents to treat SUI, considerable interest inside regenerative medicine has received the use of

stem cells to functionally regenerate this structure in cases with SUI secondary to urethral sphincter deficiency. Stem cell therapy

(SCT) has been used to regenerate injured tissues and has been investigated in several diseases [3-5]. Differently to bulking agents,

the aim of regenerative approaches with SCs is to repair the damaged components of the sphincter. The goal is to obtain a good

integration of the bioengineered cells into the tissue so to obtain a functional organ [3-5] . A prospective multicenter and

multidisciplinary in vitro and on mice study as proposed from our project, will have a high scientific impact in the international

literature, possible publication on high IF journals (Lancet Journal), rapid diffusion through international media and congresses. Our

study design and methods involved are able to produce results statistically and clinically significant and it represent a first step to

continue with a clinical trial on human patients.

The definition of a safe and standardized protocol to obtain, prepare and delivery autologous MDSC or ADSC for injection in the

human external urethral sphincter of SUI patients has the potential to produce a high technological and economic impact. Our

two-step procedure could define a reproducible and effective technology :to prepare MDSC or ADSC suspension, to protect the

suspension with synthetic scaffold so to reduce inflammatory reactions and to consent a gradual delivery of cells, to effectively inject

into the sphincter, to distribute it homogeneously, to guide and verify position of cells. Also the definition and verification of the

technology involved in tracking MDSC and ADSC integration into the sphincter so to follow their regenerative and functional potential

could have a high impact. The definition of all these steps in vitro and in vivo, will consent to develop a standardize procedure for the

human protocol. Statistically and clinically significant results using regenerative medicine and MDSC injection to obtain a long term

improvement or resolution of SIU, has the potential for a high economic impact. It may replace synthetic bulking agents for SUI

treatment, at now related to modest results as underlined by international guidelines. Interest from industry could be also relevant.