WO2021099665A1

WO2021099665A1 - Placental decidua parietalis stem cells for use in chronic stress urinary incontinence

Info

Publication number: WO2021099665A1
Application number: PCT/ES2020/070718
Authority: WO
Inventors: Ana Isabel FLORES DE LA CAL; Paz DE LA TORRE MERINO; María Jesús PÉREZ LORENZO; Álvaro ALCÁZAR GARRIDO; Eloy MUÑOZ GÁLLIGO; Ana Rosa MASERO CASASOLA; María Del Carmen GUTIÉRREZ VÉLEZ; José MEDINA POLO; Jesús Alfredo GRANDE GARCÍA; Alicia GARCÍA GARCÍA-PORRERO
Original assignee: Fundación Para La Investigación Biomédica Del Hospital 12 De Octubre
Priority date: 2019-11-22
Filing date: 2020-11-19
Publication date: 2021-05-27
Also published as: ES2827849A1; ES2827849B2

Abstract

The present invention relates to stem cells from the placental decidua parietalis (DMSCs) for use in the treatment of chronic stress urinary incontinence in a subject. This invention also relates to a pharmaceutical composition comprising said DMSCs for the same use, and the kit and device comprising same.

Description

Placenta decidua parietalis stem cells for use in chronic stress urinary incontinence

This application claims the priority of the Spanish patent application P201931035 filed on November 22, 2019.

DESCRIPTION

The present invention relates to placental decidua parietalis stem cells for use in the treatment of chronic stress urinary incontinence. Said method is useful in the regeneration of the supporting tissue that surrounds the urethra, which, for example, is damaged after pregnancy or childbirth, making it useful in the field of medicine.

STATE OF THE ART

Urinary incontinence (UI) is the involuntary loss of urine. There are several types of UI, including stress or stress UI (SUI) associated with exertion that causes increased abdominal pressure (for example, coughing, sneezing, laughing, running, or even walking). It is estimated that, in approximately 50% of women with urinary incontinence, their main symptom is SUI (Deng DM 2011 Med. Clin. N. Am. 95: 101-109).

SUI is due to sphincter deficiency and urethral hypermobility due to insufficient support of the pelvic muscles and the connective tissue surrounding the urethra and bladder neck, which affects both women and men. SUI is more common in women and increases with age, lack of estrogen, obesity, and multiple births. In women who have had children, the mechanism that gives rise to incontinence is usually hyperdistention of the pelvic floor tissues during pregnancy and childbirth, mainly during the latter, mainly due to the passage of the fetal head (Deng DM 2011 Med. Clin. N. Am. 95: 101-109). In men, SUI cases occur after prostate surgery or radiation (Juarranz M et al. Aten. Primaria 2002 30 (5): 323-332).

The quality of life of patients with SUI is compromised. Between the SUI treatments can be found, for example, non-invasive methods such as pelvic floor strengthening exercises (i.e., pelvic floor rehabilitation, for example, Kegel exercises, physical therapy), drug treatment (for example, alpha-adrenergic agonists, duloxetine) and invasive treatments such as injection of fillers (eg, polytetrafluoroethylene, bovine collagen, or silicone particles) and surgery (eg, suburethral bands). Commonly used methods for SUI do not permanently correct incontinence. In addition, the effectiveness of non-invasive techniques, as in the case of pelvic floor strengthening exercises, depends on the patient's motivation and compliance with them. In the case of drug treatments, efficacy depends on tolerance to side effects. If early SUI cases are not resolved, they tend to become chronic. In chronic cases, surgery is the method that is usually used, however, this invasive technique usually causes complications, for example, adhesions, and generally fails within a few years, so patients must undergo subsequent surgeries. , which do not usually have a high success rate (Spanish Society of Gynecology and Obstetrics (SEGO). Diagnosis of urinary incontinence. Prog. Obstet. Ginecol. 2019; 62 (1): 79-91; Deng DM 2011 Med. Clin N. Am. 95: 101-109; and Puyol M etal. 2009 Arch. Esp. Urol. 62: 882-888).

UI caused by neurogenic bladder (which appears, for example, after strokes, multiple sclerosis and spinal cord injuries) presents symptoms similar to SUI, however, in UI caused by neurogenic bladder, the pudendal nerve is affected and the clinical picture It is different from SUI, since the patient generally complains of overflow incontinence where there is no contraction of the detrusor muscle for emptying and there are constant losses of urine in relation to an overfilling of the bladder and not due to primary sphincter failure. However, in SUI due to pelvic floor weakness, for example, after pregnancy or after childbirth, it is not associated with alterations in detrusor contraction or bladder capacity, but is due to hyperdistention of the tissues of the pelvic floor and muscle damage to the pelvic support systems. SUI due to pelvic floor weakness is not due to deep neurological damage, as occurs in UI caused by neurogenic bladder due to pudendal nerve transection or by central nerve injuries; which gives rise to a totally different clinic (Blok B, et al. European Association of Urology (EAU) Guidelines on Neuro-Urology European Association of Urology 2018). Therefore, SUI due to pelvic floor weakness is produced by a primary pelvic injury injury, it is not secondary to nerve damage. The differences between neurogenic bladder UI and SUI are such that even in the study of the neurogenic bladder, another animal model is used (by denervation of nerves, for example, the pudendal nerve, which causes complete bladder dysfunction), while that in the study of SUI the model of vaginal dilation (which produces a dysfunction of the urethral closure) is used (Hijaz A et al. 2008 The Journal of Urology 179 (6): 21032110; and Koike et al. 2013 International Journal of Urology 20.1 : 64-71). The treatment of both types of UI is therefore different, since neurogenic bladder UI requires regeneration of the nerve, while in the treatment of SUI what is required is the regeneration of supporting tissues. According to the SEGO, neurogenic incontinence has a different surgical treatment protocol. Neurogenic bladder UI and SUI are different pathologies.

From what is known in the state of the art, there is still a need to find a method that achieves an effective long-term treatment of chronic SUI, thus avoiding subjecting the patient to invasive procedures and their complications.

EXPLANATION OF THE INVENTION

The inventors have developed a tool that allows the treatment of chronic stress urinary incontinence (SUI). By using stem cells from the decidua parietalis of the placenta (DMSCs), researchers have been able to regenerate those structures of the supporting tissue that surrounds the urethra. Therefore, the passive closure mechanisms of the urethra have been regenerated thanks to the connective support, which are damaged, for example, in childbirth.

The inventors have shown in in vitro experiments that human DMSCs are capable of migrating towards cells of the suburethral mucosa belonging to patients with SUI and this migration is significantly greater than the migration of DMSCs towards cells of the suburethral mucosa belonging to patients without damage. on the supporting tissue of the urethra (see example 2.1, figure 1). The DMSCs of the invention therefore migrate towards suburethral mucosal damage.

Furthermore, they have shown that cells of the suburethral mucosa of patients with SUI proliferate significantly more in vitro in the presence of DMSCs than cells of the suburethral mucosa of patients without damage to that connective tissue (see example 2.2, figure 2). The researchers believe that this result indicates that it is the mucosal cells themselves that perform the function of regenerating damaged tissue when they are in the presence of DMSCs, without the involvement of DMSCs differentiation, probably due to the release of factors by the cells. DMSCs.

In in vivo SUI experiments with a vaginal dilation model of SUI, the inventors have shown that DMSCs injected into the suburethral mucosa reverse SUI (see example 3), both acute SUI (Figure 3), and chronic SUI ( figure 4). In the case of chronic SUI, the pressure to be exerted to escape is increased (Fig. 4, 5A, 5B), increasing the pressure necessary to overflow at maximum filling in each animal (Fig. 6).

With imaging techniques the researchers were able to visualize the DMSCs in the urethra region even 2 or 3 weeks after their injection into the suburethral mucosa in the in vivo animal model (see figure 7).

In the present invention, through the use of DMSCs, both structural and functional regeneration of the urethra of chronic SUI patients is achieved.

Therefore, the first aspect of the invention refers to stem cells of the decidua parietalis of the placenta (DMSCs) for use in the treatment of chronic stress urinary incontinence in a subject, characterized in that said DMSCs express the CD44 proteins , CD90, CD117, CD73, CD29, CD13, CD105, Oct-4, Rex1, GATA-4 and HLA class I (HLA-ABC); and, in addition, they do not express the proteins CD34, CD45, CD133, BCRP1, STRO-1, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, HLA class II (HLA-DR), CD40L , CD80 and CD86. In particular the first aspect of the invention, optionally in combination with any particular embodiment provided below, relates to placental decidua parietalis stem cells (DMSCs) for use in treating chronic stress urinary incontinence in a subject, characterized in that said DMSCs cells express the proteins CD44, CD90, CD117, CD73, CD29, CD13, CD105, Oct-4, Rex1, GATA-4 and HLA class I (HLA-ABC); and, in addition, they do not express the proteins CD34, CD45, CD133, BCRP1, STRO-1, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, HLA class II (HLA-DR), CD40L , CD80 and CD86; and characterized in that the DMSCs are repeatedly administered on at least two occasions to the subject.

In the present invention, DMSCs are used as an active ingredient.

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the DMSCs can be used after they have been obtained and characterized or, alternatively, they can be frozen after obtaining and characterizing and used after thawing or culturing them after thawing for later use, following protocols known to those skilled in the art.

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, SUI is caused by deficit of the urethral sphincter.

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the treatment of SUI is carried out by regeneration of the connective tissue surrounding the urethra or bladder, or, alternatively, by regeneration of the urethral sphincter, or, by regenerating both.

The decidua is the component of the placenta of maternal origin that originates from the endometrium and contains stem cells of mesodermal origin, called decidua mesenchymal stem cells (“Decidua-derived Mesenchymal Stem Cell”, DMSC). The DMSCs of the present invention are characterized by markers that are described in Macías MI et al. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9- 495. e23.

Stress urinary incontinence (SUI) in the present invention is a SUI that occurs in a subject with pelvic floor weakness, for example, by loss of connective tissue and / or smooth muscle surrounding the urethra and that entails a deficit of the sphincter of the urethra and / or urethral hypermobility.

In the present invention, chronic SUI is understood to be that SUI that persists after one year of delivery or pregnancy or post-surgery or that which, appearing at any time, is one year or more in duration.

In the present invention, acute SUI is understood as that SUI that appears after childbirth or pregnancy or post-surgery or at any time and that lasts less than one year. In the case of SUI after childbirth, acute SUI is considered to occur when three months have passed after it but is not longer than a year in duration, since before it is considered that incontinence is normal according to experts. Once SUI exceeds the duration of one year, it is considered chronic SUI.

In the present invention, "urethral sphincter deficit" is understood as the failure in the function of the urethral sphincter (meaning "urethral sphincter" is the muscle that surrounds the urethra) that causes the loss of urine and the appearance of SUI. Urethral sphincter deficit, for example, can be caused by a deficit of connective tissue and / or by the presence of cells with a myofibroblast phenotype that are not capable of exerting a correct contraction.

In the present invention, patients do not have a neurological pathology, for example, in the pudendal nerve, which is the cause of incontinence.

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the subject does not have any nerve affected (for example, damaged) that induces UI, for example, the nerve is not affected pudendal, that is, it has a functional pudendal nerve. In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the DMSCs are administered locally in the urethra, for example, in the suburethral mucosa, intravenously, or vaginally. .

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the DMSCs are administered through local injection into the suburethral mucosa, for example, in at least two locations.

Since the treatment of SUI using the DMSCs of the present invention is not aggressive for the patient, it can be repeated as many times as necessary.

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the DMSCs are administered at least twice to the subject; for example, two, three, four, five, six, seven, eight, nine, or ten times to the subject.

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, when the DMSCs are administered on at least two occasions, between each administration of DMSCs the SUI is evaluated to decide whether the treatment has been cash or if the subject needs a new administration.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, when SUI is evaluated to decide whether the treatment has been effective or whether the subject needs a new administration, the absence of SUI is indicative that DMSC therapy has been effective and does not require re-administration of DMSCs.

Methods for evaluating whether DMSCs have been effective are known to the expert, for example: using recognized questionnaires that measure incontinence, for example, described in “Spanish Society of Gynecology and Obstetrics (SEGO). Diagnosis of urinary incontinence. Prog. Obstet. Ginecol 2019; 62 (1): 79-91 ".

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the DMSCs are obtained from the placenta of a mammal, for example, a human.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the DMSCs are obtained from human placenta.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the DMSCs are obtained from a postpartum placenta, for example from a postpartum human placenta.

The methods for obtaining the DMSCs are those described, for example, in Macías MI et al. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9-495.e23.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the DMSCs are used in combination with a drug, therefore they are used in combination therapy with a drug; for example, where the drug is one or more serotonin and norepinephrine reuptake inhibitors, or one or more alpha adrenergic agonists, or one or more estrogens, or any combination thereof.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the serotonin and norepinephrine reuptake inhibitor is duloxetine (eg Cymbalta®).

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or Below, the alpha adrenergic agonist is ephedrine, pseudoephedrine, phenylpropanolamine, or any of their combinations.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the estrogens are estrogens for local application or estrogens administered orally, or a combination of both; for example, promestriene (for example, Colpotrofin®), estriol (for example, Blissel®), estradiol hemihydrate (for example, Vagifem®), dehydroepiandrosterone (for example, Prasterone®), or any combination thereof.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, when the DMSCs are used in combination with a drug, it is administered before and / or after the administration of the DMSCs.

In a particular embodiment of the first aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the DMSCs are used in combination with pelvic floor rehabilitation, therefore they are used in combination therapy with pelvic floor rehabilitation of the patient, for example, in combination with the performance of Kegel exercises (contraction exercises of the pubococcygeus muscle) before or after receiving treatment with DMSCs.

Pelvic floor rehabilitation is performed according to standard methods known to those skilled in the art.

The first aspect of the invention can alternatively be formulated as the use of DMSCs for the preparation of a drug for the treatment of chronic stress urinary incontinence in a subject, characterized in that said DMSCs cells express the proteins CD44, CD90, CD117, CD73, CD29, CD13, CD105, Oct-4, Rex1, GATA-4 and HLA class I (HLA-ABC); and, in addition, they do not express the CD34, CD45, CD133, BCRP1, STRO-1, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, HLA class II proteins (HLA-DR), CD40L, CD80 and CD86. In a particular embodiment, the DMSCs are administered repeatedly on at least two occasions to the subject.

The first aspect of the invention can alternatively be formulated as the method of treating chronic SUI comprising the step of administering a therapeutically effective amount of DMSCs in a subject with chronic SUI, where said DMSCs cells express the proteins CD44, CD90, CD117, CD73, CD29, CD13, CD105, Oct-4, Rex1, GATA-4 and HLA class I (HLA-ABC); and, in addition, they do not express the proteins CD34, CD45, CD133, BCRP1, STRO-1, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, HLA class II (HLA-DR), CD40L , CD80 and CD86. In a particular embodiment, the DMSCs are administered repeatedly on at least two occasions to the subject.

A second aspect of the invention refers to a pharmaceutical composition characterized by comprising a therapeutically effective amount of the DMSCs of the first aspect of the invention and which also comprises one or more pharmaceutically acceptable excipients or vehicles, for use in the treatment of urinary incontinence. of chronic exertion in a subject.

The term "therapeutically effective amount" as used herein refers to the amount of the DMSCs that, when administered, is sufficient to treat or alleviate to some degree one or more symptoms of chronic SUI in a subject. The specific dose of DMSCs administered according to the invention will be determined by the specific circumstances of the case, including the route of administration, and the severity of the disease to be treated, among others.

The compositions of the present invention can be prepared by methods well known in the state of the art. The person skilled in the art can determine the excipients and / or vehicles, and suitable amounts to use, depending on the formulation to be prepared.

The term "excipient" refers to a substance that assists in the absorption of the DMSCs of the invention or the pharmaceutical composition of the invention or stabilizes said cells or pharmaceutical composition or assists in the preparation of the pharmaceutical composition. Thus, excipients could have the function of maintaining the united ingredients such as starches, sugars or celluloses, coloring function, protection function of the medicine such as for example to isolate it from air and / or humidity, filling function of a tablet, capsule or any other form of presentation, without excluding other types of excipients not mentioned in this paragraph.

The term "pharmacologically acceptable" refers to the compound to which it refers is allowed and evaluated so that it does not cause harm to the organism to which it is administered. In a particular embodiment of the second aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the pharmaceutical composition further comprises one or more serotonin and norepinephrine reuptake inhibitors, or one or more alpha adrenergic agonists , or one or more estrogens, or any of their combinations.

All the particular embodiments of the first aspect of the invention also form part of the second aspect of the invention.

A third aspect of the invention refers to a kit comprising the DMSCs defined in the first aspect of the invention or the pharmaceutical composition defined in the second aspect of the invention and also comprising one or more serotonin and norepinephrine reuptake inhibitors , or one or more estrogens, or one or more alpha adrenergic antagonists, or any combination thereof. In a particular embodiment of the third aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the serotonin and norepinephrine reuptake inhibitor is duloxetine.

In a particular embodiment of the third aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the alpha adrenergic agonist is ephedrine, pseudoephedrine, phenylpropanolamine, or any of their combinations. In a particular embodiment of the third aspect of the invention, optionally in combination with any of the particular embodiments provided above or below, the estrogens are locally applied estrogens or orally administered estrogens or a combination of both; for example, promestriene (for example, Colpotrofin®), estriol (for example, Blissel®), estradiol hemihydrate (for example, Vagifem®), dehydroepiandrosterone (for example, Prasterone®), or any combination thereof.

The kit of the third aspect of the invention may contain instructions for carrying out the use of the first aspect of the invention.

All the particular embodiments of the first and second aspects of the invention also form part of the third aspect of the invention.

A fourth aspect of the invention refers to the use of a kit comprising the DMSCs defined in the first aspect of the invention or the pharmaceutical composition defined in the second aspect of the invention or of the kit of the third aspect of the invention for the treatment of Chronic stress urinary incontinence of a subject.

In a particular embodiment of the third or fourth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the kit further comprises a device comprising the DMSCs, for example, a pre-filled syringe with the DMSCs defined in the first aspect of the invention or the pharmaceutical composition defined in the second aspect of the invention.

All the particular embodiments of the first, second and third aspects of the invention also form part of the fourth aspect of the invention.

A fifth aspect of the invention refers to a device comprising the DMSCs defined in the first aspect of the invention or the pharmaceutical composition defined in the second aspect of the invention. In a particular embodiment of the fifth aspect of the invention, optionally in combination with any particular embodiment provided above or below, the device is a syringe.

All embodiments of the first, second, third or fourth aspects of the invention are particular embodiments of the fifth aspect of the invention.

In a particular embodiment of the first, second, third, fourth or fifth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the DMSCs may be marked for display.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the use is carried out in combination with the subject's imaging to observe the correct administration of the DMSCs and / or or its distribution in the suburethral mucosa and / or in the surrounding tissue.

In a particular embodiment of the first, second, third, fourth or fifth aspects of the invention, optionally in combination with any particular embodiment provided above or below, when duloxetine is administered, it is administered to the subject in an amount of 40-80 mg per day for at least 2 weeks, for example 2-4 weeks.

In the present invention, the terms "subject", "patient" or "individual" are used interchangeably.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the subject presenting chronic SUI is a human. The patient can be of any age, gender, or race.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any of the particular embodiments provided above or below, the subject is a woman; for example, is a woman who It is selected from the group consisting of: nulliparous, primiparous, multiparous, nulliparous, primiparous, and multiparous.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the subject is a nulliparous and nulliparous (nulligravid) woman; or, alternatively, nulliparous and primigravida (primigravida) (for example, the woman who has had one or more children by caesarean section); or, alternatively, nulliparous and multigesta (multigravida) (for example, the woman who has had one or more children through two or more caesarean sections); or, alternatively, the woman who has had one or more childbirth (multiparous, for example, secundiparous, tertiary, quartiparous, quintiparous, sextiparous, septiparous, octiparous, noniparous or deciparous).

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any of the particular embodiments provided above or below, the subject is a menopausal woman.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any of the particular embodiments provided above or below, the subject is a woman with chronic SUI who does not have any nerve affected that being damaged induces UI. , for example, the pudendal nerve is not affected.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the subject is a woman who has previously received another treatment for SUI but has not been effective, for example , one or more surgeries.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any of the particular embodiments provided above or below, the subject is a man presenting with deficit in the urethral sphincter; for example, it is a man who has damage to the urethral sphincter from having previously undergone prostate surgery, for example, to remove a prostate tumor. In a particular embodiment of the first, second, third or fourth aspects of the invention, optionally in combination with any of the particular embodiments provided above or below, the DMSCs are obtained from the placenta of an individual other than the recipient subject of the DMSCs; that is, it is an allogeneic use.

In a particular embodiment of the first, second or fourth aspects of the invention, optionally in combination with any of the particular embodiments provided above or below, the DMSCs are obtained from the placenta of a woman who is the recipient subject of the DMSCs; that is, it is an autologous use.

In a particular embodiment of the first, second, third or fourth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the subject is under 65 years of age; or, alternatively, the subject is 65 years of age or older.

In a particular embodiment of the first, second or fourth aspects of the invention, the use of the invention further comprises the steps of (i) compiling the information resulting from using the DMSCs, or the pharmaceutical composition or the kit, and (ii) storing the information on a data carrier.

In the sense of the invention, a "data carrier" should be understood as any medium that contains information data on the use of the first, second or fourth aspects of the invention or of the patient's response to said uses, such as paper. The support can also be any entity or device capable of transporting the data. For example, the data carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, a DVD, or a magnetic recording medium, for example a hard disk. Furthermore, the carrier can be a transmissible carrier such as an electrical or optical signal, which can be transmitted through an electrical or optical signal, cable, or by radio or other means. When the data is incorporated into a signal that can be carried directly by a cable or other device or medium, the support can be constituted by said cable or other device or medium. Other operators are related to USB devices and computer files. Examples of suitable data carriers are paper, CD, USB, computer, PC files, or sound record with the same information. For the purposes of the present invention, any given range includes the lower and upper end points of the range. All terms used in this application, unless otherwise indicated, will be understood in their ordinary meaning as known in the art. Other more specific definitions of certain terms used in the present application are those set forth herein and are intended to be applied uniformly throughout the description and in the claims, unless an expressly stated definition otherwise provides a broader definition. The definitions given herein are included for the purpose of understanding and are expected to apply throughout the description, claims, and drawings.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. Furthermore, the word "comprises" includes the case "consists of". For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, shows the in vitro migration of DMSCs in a migration chamber when they are in the presence of cells from the suburethral mucosa of patients with SUI ("SUI") or cells from the mucosa of patients without damage to the supporting soil tissue. pelvic (patients with uterine prolapse and without SUI, “POP”).

FIG. 2, shows a comparison between the in vitro proliferation of cells of the suburethral mucosa of patients with SUI (“SUI”) and of cells of the suburethral mucosa of patients without damage to the supporting tissue of the pelvic floor (patients with uterine prolapse and without SUI, "POP"), when they were in the presence of DMSCs. FIG. 3, comparison of the pressure required to escape between the control group (HBSS) and the group of rats with acute damage (acute SUI) that received two doses of 2x10 ⁶ DMSCs injected into the suburethral mucosa, measured with 200 microliters of filling. Data at 1, 3, 5 and 6 weeks after the first administration of the DMSCs.

FIG. 4, comparison of the pressure necessary to escape between the control group (HBSS) and the group of rats with chronic damage (chronic SUI) that received two doses of 2x10 ⁶ DMSCs injected into the suburethral mucosa, measured with 200 microliters of filling. Data at 1, 2, 3 and 4 weeks after the first administration of the DMSCs.

FIG. 5, comparison of the pressure necessary to escape between the control group (HBSS) and the group of rats with chronic damage (chronic SUI) that received two doses of 2x10 ⁶ DMSCs injected into the suburethral mucosa, measured with 100 (A) or 200 (B) microliters of filling, after repeated application of the treatment. Data at 11 weeks after the first administration of the DMSCs.

FIG. 6, comparison of the pressure necessary to escape between the control group (HBSS) and the group of rats with chronic damage (chronic SUI) that received two doses of 2x10 ⁶ DMSCs injected into the suburethral mucosa, when measured with the maximum volume that each rat had, after repeated application of the treatment. Data at 11 weeks after the first administration of the DMSCs.

FIG. 7, Visualization of human DMSCs in vivo stained and injected into a post-damage rat at different times (days). Panel "A", rats that received treatment with DMSCs; panel "B", rats that received the control (HBSS).

EXAMPLES 1. Obtaining and characterizing mesenchymal stem cells from the decidua parietalis of the placenta:

The stem cells of the decidua parietalis were obtained and characterized following the protocols described in the document Macías MI et al. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9-495.e23. The materials and methods used are briefly indicated below:

Material and methods:

Obtaining the placentas:

Human placentas from term pregnancies were obtained by informed consent approved by the Ethics Committee for Clinical Research (CEIC) of the Hospital Universitario 12 de Octubre (Madrid, Spain).

Obtaining cells from the amniotic sac:

The placentas were processed immediately after delivery. To obtain the cells, the amniotic sac was mechanically separated from the chorionic villi with the help of forceps and scissors and the chorionic villi was discarded. The membranes of the amniotic sac were washed with 1X Phosphate Buffer Saline (PBS) and the cells were obtained by two digestions of thirty minutes each with 0.05% trypsin-EDTA. The cells obtained were seeded at a density of 1.16 x 10 ⁵ cells / cm ² in the culture medium composed of Modified Dulbecco Essential Medium (DMEM, Lonza), 2 mM glutamine, 1 mM sodium pyruvate, 10 ng / ml of epidermal growth factor (EGF, Sigma), 10% fetal bovine serum (Lonza), 1% antibiotics (Penicillin-Streptomycin 10,000 Units, Lonza), 1% non-essential amino acids (Lonza) and 55 mM of b -mercaptoethanol (Sigma) and kept in an incubator at 37 ° C and 5% C0 ₂ .

Crop maintenance:

Non-adherent cells were removed five days after the start of the cultures, by aspiration of the culture medium and washing with 1X PBS (Lonza). The culture medium was replaced every four days until the cells reached 90-100% confluence. The cells were detached from the culture plate by digestion with trypsin-Versene (five minutes at 37 ° C and 5% CO2) and seeded at a concentration of 4-5 x 10 ⁴ cells / cm ² . The DMSCs did not differentiate, they were they were kept in complete DMEM medium until use or frozen for later use (freezing according to standard procedures, in early passages, that is, at 2, 3 or 4 passages). When cells were frozen, they were used after thawing and expansion. DMSCs were generally used in passage 6-10.

Study of the maternal-fetal origin:

The fetal or maternal origin of the DMSCs was analyzed using two different techniques from cells obtained from three placentas in which the child born was male; through the fluorescent in situ hybridization technique and through the study of short tandem repeats. a) - Fluorescent in situ hybridization (FISH):

The Poseidon FISH Kit (Kreatech) was used to carry out this study. According to the manufacturer's instructions, the cells were grown adhered to a sterile coverslip and once 70% confluence was reached in the culture, they were washed with PBS for ten minutes at 37 ° C and fixed with Carnoy's Fixative (Methanol acetic 3: 1) cool for ten minutes at room temperature. Subsequently, they were permeabilized for 15 minutes at 37 ° C in permeabilization buffer (0.5% Igepal CA-630 (Sigma) diluted in 2X Sodium Citrate Saline (2X SSC) and dehydrated by incubation in a series of ethanol at 70%, 85% and 100% for one minute each. Cells were incubated with 10 pL of the fluorescent probes (Poseidon repeat-free XY robes, Kreatech) at 75 ° C for ten minutes to denature both deoxyribonucleic acid (DNA ) cells as the probes and were incubated at 37 ° C in a humid chamber overnight to allow hybridization of the probe with the cellular deoxyribonucleic acid (DNA). The already hybridized cells were then washed in the presence of buffer of Wash I (0.3% Igepal in 0.4% SSC, contained in the Poseidon FISH Kit, Kreatech) for two minutes at room temperature and with shaking. The sample was then washed in this same buffer for two minutes at 72 ° C without stirring. Finally, the sample was washed in Wash Buffer II (0.1% Igepal in 2X SSC, contained in the Poseidon FISH kit, Kreatech) for one minute at room temperature and without shaking. The The sample was dehydrated again, using a series of 70%, 85% and 100% ethanol for one minute each and dried at room temperature. Cell nuclei were stained with 0.2 pg / mL of 4 ', 6'-diamidino-2-phenylindole (DAPI, Fluka) for one minute at room temperature and darkness. Finally, the cells were visualized by fluorescence microscopy. b) - Study of short tandem repeats (STRs).

This study was performed using the quantitative fluorescent polymerase chain reaction (QF-PCR) technique with the Aneufast ™ QF-PCR kit (Molgentix). This technique made it possible to detect the presence or absence of a certain chromosomal fragment through the use of fluorescent probes. Following the manufacturer's instructions, a multiple study of the markers AMXY, SRY, X22, DXYS218, HPRT was performed, as described in Macías MI et al. 2010.

The reaction mixture was carried out according to the instructions indicated by the manufacturer of the Aneufast ™ QF-PCR kit and the thermal cycle was carried out on a 3100 genetic analyzer, with technology based on capillary electrophoresis (Applied Biosystems) according to the published protocol. by Mann et al. in 2001 (Mann K, et al. 2001 The Lancet 358: 1057-1061).

Population dynamics:

To measure the number of cells that the placenta was capable of producing, that is, to measure the number of times it doubled and the time it took to do so, a population dynamics study was carried out.

The growth dynamics of the cultures (n = 9) was determined by counting the cells in a Neubauer chamber at each passage. The number of cell duplications was calculated using the formula: N _t = No x 2 ^d , where "N _t " was the number of cells at time t, "No" was the number of initial cells and "d" was the number of divisions. cells that had occurred throughout the culture (at time t). The data obtained from nine different placentas were analyzed with the Microsoft Excel® computer program to obtain the mean and standard deviation of the parameters generation time, culture time, number of passages and number of cell divisions and the graphs were constructed with the computer program SigmaPlot® V 11.0 (Sigma).

Study of markers of DMSCs cells:

The analysis was carried out following the protocol described in Macías MI et al. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9-495.e23. a) - Flow cytometry:

For flow cytometric analysis, cell suspensions were obtained by trypsin-Versene digestion (as indicated above). The cells were resuspended in 1X PBS with 2% horse serum at a concentration of 5x10 ⁵ cells per 100 µl of buffer. Cells were stained with antibodies specific for human markers CD45 PerCP (BD Pharmingen), CD34 FITC, CD133 / 1 PE (Miltenyi), CD105 FITC (Serotec), BCRP1 FITC (Millipore), CD73 PE, CD29 PE, CD44 FITC , CD117 PE, CD90 FITC and CD13 PE (BD Pharmingen), HLA-ABC FITC (MHC class I), HLA-DR FITC (MHC class II), CD40 FITC, CD80 (B7-1)

FITC, CD86 (B7-2) PE, SSEA-1 PE, SSEA-3 PE, SSEA-4 PE, TRA-1-60 PE and TRA-1- 81 PE (eBioscience). To calculate and correct the nonspecific binding of the antibodies, the control isotype analysis was performed for each marker. The isotypes examined were Mouse IgG1 FITC, Mouse IgG2a FITC (Serotec), Mouse IgG1 PE, Mouse IgG1 PerCP, Mouse IgG2a PE (Becton Dickinson), Mouse IgG3 FITC and Mouse IgM PE (eBioscience). Antibodies were used at a 1:10 or 1:20 concentration, according to the manufacturer's instructions and taking their control isotypes as reference. The cells were incubated in the presence of the antibodies for fifteen minutes at room temperature and in the dark. Subsequently, at least 20,000 events were analyzed for each marker, on a FACScan® flow cytometer (Becton Dickinson) equipped with the CelIQuest Pro® computer program. b) - Reverse transcription and polymerase chain reaction (RT-PCR):

It was carried out to study the expression of the Oct-4, Rex-1, GATA-4, HLA-ABC and HLA-DR.

The purification of ribonucleic acid (RNA) from the cells obtained from the placenta was carried out with the RNA isolation kit "AquaPure" (Bio-Rad) or with the RNA purification kit MasterPureTM (Ecogen) following the manufacturer's instructions. . To obtain RNA, samples both frozen at -80 ° C and fresh samples were processed once the cells had been washed two or three times with 1X PBS. After obtaining the nucleic acid, it was resuspended in 35 m \ - of TE buffer (10 mM Tris-HCl, pH = 7.5 and 1mM EDTA) and was processed immediately or stored until use at -80 ° C. The amount of RNA obtained, as well as its possible contamination with proteins and / or salts, was determined by measuring it in a ND-1000 spectrophotometer (NanoDrop, Thermo Scientific). RNA quality was determined by electrophoresis on a 1.5% agarose gel (Sigma) in 1X Tris / Acetic Acid / EDTA (TAE, Bio-Rad) buffer. EZ LoadTM Molecular Rulers (Bio-Rad) pre-stained markers were used as molecular weight standard. Agarose gels were stained with SYBR® Gold (Molecular Probes) and visualized with a ChemiDocXRS transilluminator equipped with Quantity One 1D software (Bio-Rad). The synthesis of the complementary DNA (cDNA) was carried out by the reverse transcription technique (reverse) from the purified RNA.

To study the expression of the Oct-4, Rex-1, GATA-4, HLA-ABC and HLA-DR genes, by means of RT-PCR, oligonucleotides and conditions were used as described in Macías MI et al. . 2010 Am J Obstet Gynecol 203 (5): 495.e9-495.e23. c) - Immunofluorescence:

It was used to analyze the expression of STRO-1.

The cells were removed from the culture medium, washed with 1X PBS and fixed with 10% formalin (Sigma) in 1X PBS for ten minutes at room temperature. They were then washed two or three times with 1X PBS, permeabilized with 0.5% Tween-20 in 1X PBS (PBT) for 10 minutes and incubated for two hours. in blocking solution (5% horse serum (Lonza) dissolved in 1X PBS). The cells were incubated with the primary antibody obtained in mouse against the STRO-1 protein (R&D Systems) at a 1:50 dilution overnight at 4 ^o C. The primary antibody was then removed, the cells were washed three times. times in 1X PBS for five minutes and incubated with fluorochrome-conjugated secondary antibodies (Jackson Immunoresearch) at a concentration of 1: 200 for one hour. The cells were washed three times with 1X PBS for five minutes and the nuclei were stained with 0.2 mg / mL DAPI for one minute. Finally, the cells were visualized by fluorescence microscopy on a Leica DMIL microscope.

Results:

Using FISH, analyzing the presence of the sex chromosomes X and Y, the presence of two X chromosomes was observed in all the samples analyzed in all the cells obtained from placentas in which the baby born was male (n = 3 ). This result indicated that the DMSCs obtained in the three analyzed placentas were of maternal origin.

On the other hand, the results obtained using the study of repeated tandem sequences, showed that the DMSCs presented absence of the SRY fragment added to the presence of a single expression peak for the AMXY marker and two peaks for HPRT, showing that cells obtained from the three placentas had a pattern of chromosomal fragments similar to that of female cells. The DMSCs were compared to control male and female epithelial cells. In conclusion, the chromosomal analysis of the three samples analyzed showed an expression pattern compatible with the female sex.

The growth dynamics of the DMSCs was exponential. The DMSCs reached 19.08 ± 2.68 passages in which 19.5 ± 4.32 divisions of the original population occurred with a mean generation time of 96 ± 24.72 hours. The initial mean number of adherent cells at confluence was 6.88 ± 5.23 x 10 ⁶ and the mean number of cells reached in the final population was 2.54 ± 4.97 x 10 ¹⁴ in a time of 122, 33 ± 25.62 days. Data were expressed as mean ± standard deviation. The studies carried out concluded that the cells were indeed human mesenchymal stem cells of the decidua of the placenta (DMSCs), as described in Macías MI et al. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9-495.e23.

The DMSCs expressed the markers CD44, CD90, CD117, CD73, CD29, CD13, CD105, Oct-4, Rex1, GATA-4 and HLA class I (HLA-ABC); and, in addition, they did not express the proteins CD34, CD45, CD133, BCRP1, STRO-1, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, HLA class II (HLA-DR), CD40L , CD80 and CD86.

The DMSCs obtained and characterized were used in the examples described below.

2. In vitro studies of DMSC migration and suburethral mucosal cell proliferation:

Material and methods:

In order to know if the migration of DMSCs was affected by the presence of cells of the suburethral mucosa (myofibroblasts) and to check if the cellular proliferation of cells of the suburethral mucosa was affected by the presence of DMSCs, experiments were carried out with cells of the suburethral mucosa obtained from women operated on at the 12 de Octubre hospital (Madrid, Spain) (obtained through informed consent previously approved by the Clinical Research Ethics Committee (CEIC) of the Hospital Universitario Hospital 12 de Octubre) and the DMSCs obtained at the Example 1. The results between a group of patients presenting chronic stress urinary incontinence were compared with a control group (negative control) where cells were obtained from patients who had pelvic organ prolapse (POP) but did not present urinary incontinence of effort.

Cells with fibroblast morphology were obtained from the suburethral mucosa by digestion with 0.15% collagenase type II collagenase overnight at 37 ° C.

Once the cells had been disaggregated and the fibroblasts were obtained, they were cultured in complete DMEM medium (Gibco) with fetal bovine serum (FBS) (Hyclon). The cells obtained were cultured in DMEM medium with 10% fetal serum. The experiments were carried out with the cells in similar passages, always trying to be in intermediate passages, between passage 5 and passage 9, approximately.

2.1. In vitro migration experiment:

In this case, the migration of DMSCs in the presence of cells from the suburethral mucosa of 8 patients with chronic SUI was compared to those of 7 control patients (POP). The data were obtained with at least 2 experiments for each patient, with at least 3 replicates.

The Cell Biolabs migration kit "Cyto Select ™ 24-well cell migration assay" (reference CDA-100) was used, following the manufacturer's instructions. In 24-well plates, 30,000 cells of the suburethral mucosa were seeded in the lower part (except in one well that served as a control), while in the well ("transwell") that was then placed on top, the DMSCs were seeded ( 1/5 of the cells below, that is, 6,000 DMSC cells). The "transwell" had a pore size of 8 microns, so the DMSCs could cross it and migrate to the other side. The control well without cells of the suburethral mucosa served to observe the migration of the DMSCs into an empty well. The well with the DMSCs was placed on top and after an incubation of between 16-18 hours (in DMEM medium with 10% FBS) the migration of the DMSCs towards the mucosa cells of each patient was evaluated by staining, following the instructions from the manufacturer of the kit used. The cells that had passed to the other well were stained, the dye was extracted and with a colorimetric reaction at 560 nM the migration capacity was measured.

Results of the in vitro cell migration experiment:

The results after 24 or 48 hours were similar. The results at 24 hours are shown in Figure 1.

Significant differences were observed between the migration of the DMSCs that were in the “transwells” of the wells with the cells of the mucosa of patients with SUI versus those that were in the “transwells” of the wells with the control (patients with POP), the migration being higher in the DMSCs that were in the “transwells” of the wells with the mucosa cells of patients with SUI (p = 0.0127, obtained with student's t) (see Fig. 1) . The results showed that the DMSCs migrated significantly more with the cells of SUI patients where the supporting tissue was damaged, so that the DMSCs responded to a stimulus of cell damage by migrating towards the damaged area.

2.2 In vitro proliferation experiment:

In this case, the proliferation of the cells of the suburethral mucosa of 5 patients with chronic SUI in the presence of DMSCs was compared. The proliferation of cells obtained from 5 control patients (with POP) in the presence of the DMSCs was also studied. The data were obtained with at least 3 experiments for each patient, the majority with 4 replicates.

In this case, the Invitrogen “PrestoBlue Cell Viability Reagent” kit (catalog number A13261) was used following the manufacturer's instructions.

In the 24-well plates (NUNC), 30,000 cells of the mucosa of each pathology were seeded in the bottom well (except in one well that served as a control), while in the well that was then placed on top ("transwell") The DMSCs were seeded (in a ratio of 1/5 or ½ that is 6000 or 15000 with respect to the number of cells seeded below). The "transwell" was 0.4 microns, with which the cells shared the culture medium (DMEM with 10% FBS), cytokines or any other signal that the two cell types exchange, but the cells could not pass DMSCs to the other side of the "transwell". Both cells were allowed to remain in their wells for 24 hours, so that the mucosa cells adhered to the plastic and the DMSCs to the traswells. The following day, the "transwells" were joined (they were placed on top of the well with the mucosa cells), and it was waited 24h or 48h to make the activity and cell viability measurements with the AlamarBIue ™ reagent.

Results of the in vitro cell proliferation experiment: Between the two amounts of DMSCs used, 1/5 and ½, that is 6,000 or 15,000 with respect to the number of cells seeded with mucosa, the results were similar, figure 2 shows the results obtained with 6,000 DMSCs.

The proliferation of the mucosal cells was significantly higher in the case of the patients with SUI, compared with the control (the patients with POP)

(p = 0.0181, obtained with student's t) (see figure 2) when they were in the presence of DMSCs. These results indicated that the DMSCs caused a significant increase in the cell viability of the supporting tissue that was damaged, that is, in the cells of patients with SUI, compared to the supporting cells of patients with the undamaged supporting tissue (patients with POP without SUI).

3. In vivo studies in animal model of SUI:

Material and methods:

IUE model:

To perform vaginal distention simulating damage caused at the time of delivery, Sprague-Dawley rats (225-250g) were used to which the vagina was dilated using Otis Bougie urethral dilators of sizes between 24-32 previously lubricated and under anesthesia . Subsequently, a 10Fr Foley catheter was inserted into the vagina and the balloon was inflated to 3 mL. This balloon was kept in the animals for 4 hours with anesthesia (isoflorane (AbbVie) in inhaled flow at 5% for induction of analgesia and at 2% for maintenance) and analgesia (meloxicam, 5 milligrams per milliliter, diluted 1/10 in sterile physiological saline; 200 microliters were injected, subcutaneously; Metacam®, Boehringer Ingelheim) to avoid pain after treatment, for 6 hours after damage. In addition, their constants were monitored (using a pulse oximeter to monitor the heart rate and oxygen saturation while the damage lasted), they were applied eye protection (ocular petroleum jelly) and they were injected with atropine to avoid respiratory problems due to the generation of pulmonary mucus (atropine sulfate, 1 milligram per milliliter diluted 1/10 in sterile saline; 200 microliters were injected, subcutaneously; trade house, B Braun). Stress urinary incontinence was measured by measuring the pressure of emptied.

The rats had a baseline measurement of their urine leakage pressures, before damage and after damage. When the rats were measured the minimum pressure with which they escaped (that is, an overflow of the blue-stained PBS that was introduced via a probe into the previously emptied bladder was seen) after the damage, it was observed that barely touching them was produced This overflow is then when it was concluded that they were incontinent (according to Cannon TW, et al. "Effects of vaginal distension on urethral anatomy and function." BJU Int. 2002; 90 (4): 403-407).

Transplantation of DMSCs in the animal model:

Subsequently, the DMSCs were transplanted (injected) into the rats with SUI. Two groups of rats were designed in which the transplantation was carried out, one group of rats were injected with the resuspended DMSCs in HBSS and the other group of rats were only injected with HBSS, in the first, or in the fifth week. after causing the damage. The reason for choosing two different periods was to evaluate the effect of transplantation both in rats with recent damage (which was similar to immediate postpartum damage) or with old damage (which corresponds to the UI that appears in women years after delivery. , usually after the menopause and that occurs, most often between 50 and 60 years of age).

Therefore, one week (for the evaluation of acute damage) or 5 weeks (for the evaluation of chronic damage) after causing the damage, the rats were anesthetized and the DMSCs were injected suburethrally on each side of the urethra, 2 million DMSCs in 120 microliters of HBSS directly into the damaged area (approximately half was punctured on each side of the urethra). A week after the first administration, another injection was made with the same number of cells and in the same way. The cells were administered in two consecutive weeks (once a week) to ensure that no tissue damage occurred due to the high concentration of cells and that it was well tolerated by the animal. Therefore, since 2 million cells were administered to the animal in each injection, together, a total of 4 million DMSCs were administered to each animal. Rats injected with saline serum (HBSS) were used as a control group. At least 10 animals per group (n = 10) were included to obtain data that could be statistically evaluated. In the case of the experiments with chronic damage, a total of four experiments were carried out, so that a total of 18 animals with HBSS and 20 with DMSCs were used.

In addition, the effect of DMSCs cells on chronic SUI was also evaluated when they were injected repeatedly over several weeks into 5 rats, as indicated above (2 million DMSCs in 120 microliters of HBSS were used in each administration and injected half to each side of the urethra). In this experiment, the doses of two injections were repeated in two consecutive weeks and three sets of repetitions were made, waiting two weeks between each interval of injections. That is, the baseline measurement was taken at week 0 and then in the first week the vaginal distention (damage) was made, after 5 weeks (at the sixth week of the beginning of the experiment) the cells were administered and also the following week (in the seventh week), two weeks were waited (weeks 8 and 9) and the administration was repeated in weeks 10 and 11 (second round of administration), two weeks were waited (weeks 12 and 13), administered the third round of DMSCs at weeks 14 and 15 and waited two weeks (weeks 16 and 17) to see evolution and sacrifice of the animals. Bladder pressure measurements were made each week.

The bladder was also completely emptied in the rats and the maximum volume that all rats were capable of holding in the bladder was measured.

In vivo functional analysis:

Functional regeneration was assessed after cell transplantation by the drop pressure point test (LPP, Leak Point Pressuré). The method consisted of introducing a catheter into the urethra connected to a syringe with flow pressure and a pressure translator. The bladder was filled with different amounts from 0.1 mL, or 0.2 mL of saline (stained blue with methylene blue for better visualization) to reach half its filling capacity for rats of the weight used in this study and as a last measure the maximum volume admitted until the overflow. The measurement was also carried out with the maximum volume admitted before overflowing each animal.

Gentle pressure was applied to the abdomen of the rats until a saline drip was obtained, which was recorded with the Biopac data acquisition system (Astromed). The data referred to bladder pressure without abdominal pressure. The basal pressure inside the bladder when it was empty was around 2 millimeters of mercury pressure (calibrated with a manual manometer and measured by the AstroMed Biopac apparatus). In order to obtain the data of the pressure difference in the bladder (leak pressure), the pressure value after the introduction of the saline solution was subtracted from the pressure that was applied manually until seeing when the introduced serum overflowed.

The test was repeated 3 times in each animal for several weeks and the mean of the LPP was calculated for each animal.

The results were evaluated using the "student's t" calculated with the statistical program Graph Pad.

Visualization of DMSCs in the in vivo animal model:

To see how long the cells remained in the damaged area in the in vivo animal model, DMSCs cells labeled with a fluorescent marker in the Vivotrack 680nm near infrared area (Perkin Elmer Ref: NEV12001) were used. Negative control rats were injected with HBSS.

Cell staining was performed following the manufacturer's protocol, cells were concentrated to a known volume, 25 microliters of DMEM and another 25 microliters of the dye (VIVOTRACK 680, Perkin Elmer) resuspended in water, for every 10 million cells, for 15 minutes at room temperature, two washes were performed with DMEM medium without FBS or with PBS. 2 million cells were resuspended at the end of the run in 120 microliters of HBSS. Cellular staining was checked with the Facscalibur Cytometer, to see the percentage of live and stained cells. In general, in each staining it was observed that the cells present 95% live cells and 80% stained cells. After transplantation of the labeled cells into the suburethral mucosa as indicated above, they were examined in vivo for the following 3 weeks after transplantation, using the in wVo XTREME I bioluminescence apparatus (Bruker).

3.1 Results in acutely damaged rats:

In the case of acutely damaged rats, a statistically significant difference was observed between the group treated with DMSCs and the control group in the pressure necessary for dripping (after administering 200 μl) in the fifth week post-treatment (p = 0 , 02), which also happened in the sixth week post-treatment (p = 0.03) (counted from the first week of administration of the cells); where the pressure difference required for the drip in the rats treated with DMSCs was greater than in the rats treated with the control (figure 3).

3.2 Results in chronically damaged rats

In relation to the chronic damage experiments, when the cells were applied and waited 4 weeks (after administering 200 pl), as can be seen in Figure 4, the results were very consistent and in the fourth week post-treatment (counted from the first time since the administration of the cells) a statistically significant difference was observed between the group treated with the DMSCs and the control group (p = 0.03) at four weeks post-treatment; so that the pressure required for the drip in the rats treated with DMSCs was higher than in the rats treated with the control (figure 4).

In the rats of the repeat treatment experiment, at the end of the same (11 weeks counting from the first administration of the cells, which were injected at the sixth week from the beginning of the experiment) (that is, 17 weeks from the damage, but 11 weeks from the first injection), a statistically significant difference was observed, in relation to the difference in pressure required for the drip, between the group treated with the DMSCs and the control group (p = 0.0034 after administering 100 ml; p = 0.01 after administering 200 ml) so that the The pressure required for dripping in the DMSC-treated rats was higher than in the control-treated rats (Figures 5A and 5B). The difference with the mean of the whole group and every week between both groups was also statistically significant (p = 0.005, after administering 200 µl).

In the comparison of the maximum filling of the bladder between the control group (HBSS) and the group of rats with chronic damage that received the DMSCs injected into the suburethral mucosa, after repeated application of the treatment, it was observed that the rats that received the control ( HBSS) retained much less volume (measured by the pressure difference) than those that had received the treatment with the cells (see figure 6), with a statistically significant difference of p = 0.01 at the end of the experiment (11 weeks after the first administration of DMSCs).

The maximum volume of all rats was measured during the whole process, the statistical data referred to the complete group, both control and animals with cells, during the whole process (that is, each animal had 11 measurements). It was observed that in those rats with better leak pressure data (those that received treatment with the DMSCs) the volume that they were able to retain in the bladder increased (data not shown).

3.3 Results of the localization of DMSCs cells in rats:

Using imaging techniques, the researchers were able to visualize the DMSCs in the urethra region even 2 or 3 weeks after their injection into the suburethral mucosa in the in vivo animal model (see figure 7, where this localization is appreciated up to 10 days after administration of the cells).

LIST OF APPOINTMENTS

Deng DM 2011 Med. Clin. N. Am. 95: 101-109.

Juarranz M etal. Aten. Elementary 2002 30 (5): 323-332.

Spanish Society of Gynecology and Obstetrics. Incontinence diagnosis urinary. Prog. Obstet. Ginecol. 2019; 62 (1): 79-91.

Puyol M etal. 2009 Arch. Esp. Urol. 62: 882-888. Blok B. et al. European Association of Urology (EAU) Guidelines on Neuro-Urology European Association of Urology 2018.

Daughter A etal. 2008 The Journal of Urology 179 (6): 21032110. Koike et al. 2013 International Journal of Urology 20.1: 64-71.

Macías MI etal. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9-495.e23.

Mann K, et al. 2001 The Lancet 358: 1057-1061.

Cannon TW, et al. BJU Int. 2002; 90 (4): 403-407.

Claims

1. Placenta decidua parietalis stem cells (DMSCs) for use in the treatment of chronic stress urinary incontinence (SUI) in a subject, characterized in that said DMSCs express the CD44 proteins,

CD90, CD117, CD73, CD29, CD13, CD105, Oct-4, Rex1, GATA-4 and HLA class I (HLA-ABC); and, in addition, they do not express the proteins CD34, CD45, CD133, BCRP1, STRO-1, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, HLA class II (HLA-DR), CD40L , CD80 and CD86; and characterized in that the DMSCs are repeatedly administered on at least two occasions to the subject.

2. DMSCs for use according to claim 1 wherein chronic SUI is caused by urethral sphincter deficit.

3. DMSCs for use according to any of claims 1 or 2, wherein the treatment of chronic SUI is performed by regeneration of the connective tissue surrounding the urethra or bladder, or, alternatively, by regeneration of the urethral sphincter, or, by regenerating both.

4. DMSCs for use according to any of claims 1 to 3, characterized in that the DMSCs are administered in the suburethral mucosa, intravenously or vaginally.

The DMSCs for use according to claim 4, characterized in that the DMSCs are administered through local injection into the suburethral mucosa in at least two locations.

6. The DMSCs for use according to any of claims 1 to 5, characterized in that between each administration of DMSCs the SUI is evaluated to decide if the treatment has been effective or if the subject needs a new administration.

7. DMSCs for use according to any of claims 1 to 6, wherein the DMSCs are obtained from human placenta.

8. DMSCs for use according to any of claims 1 to 7 in combination therapy with a drug, wherein the drug is one or more serotonin and norepinephrine reuptake inhibitors, or one or more alpha adrenergic agonists, or one or more more estrogens, or any combination thereof; and / or in combination with pelvic floor rehabilitation.

The DMSCs for use according to claim 8, wherein the serotonin and norepinephrine reuptake inhibitor is duloxetine.

10. DMSCs for use according to any of claims 8 or 9, wherein the alpha adrenergic agonist is ephedrine, pseudoephedrine, phenylpropanolamine or any of their combinations.

The DMSCs for use according to any one of claims 8 to 10, wherein the estrogen is promestriene, estriol, estradiol hemihydrate, dehydroepiandrosterone, or any of their combinations.

12. The DMSCs for use according to any one of claims 1 to 11, wherein the subject is a female.

The DMSCs for use according to claim 12, wherein the subject is a female who is selected from the group consisting of: nulliparous, primiparous, multiparous, nulliparous, primiparous and multiparous.

The DMSCs for use according to any of claims 12 or 13, wherein the subject is a menopausal woman.

15. The DMSCs for use according to any one of claims 1 to 11, wherein the subject is a man with a deficit in the urethral sphincter.

16. The DMSCs for use according to claim 15, wherein the subject is a man who has damage to the urethral sphincter due to having previously undergone prostate surgery.

17. DMSCs for use according to any one of claims 1 to 16, wherein the DMSCs are obtained from the placenta of an individual other than the recipient subject of the DMSCs.

18. DMSCs for use according to any of claims 1 to 14, wherein the DMSCs are obtained from the placenta of a woman who is the recipient subject of the DMSCs.

19. DMSCs for use according to any of claims 1 to 18, wherein the subject has a functional pudendal nerve.

20. A pharmaceutical composition characterized by comprising a therapeutically effective amount of the DMSCs defined in any of claims 1 to 19 and further comprising one or more pharmaceutically acceptable excipients or carriers, for use in the treatment of chronic SUI in a subject.

21. Kit comprising the DMSCs defined in any of claims 1 to 19 or the pharmaceutical composition defined in claim 20 and further comprising one or more serotonin and norepinephrine reuptake inhibitors, or one or more estrogens, or one or more plus alpha adrenergic antagonists, or any combination thereof.

22. The kit according to claim 21, wherein the serotonin and norepinephrine reuptake inhibitor is duloxetine.

23. The kit according to any of claims 21 or 22, wherein the alpha adrenergic agonist is ephedrine, pseudoephedrine, phenylpropanolamine, or any of their combinations.

24. The kit according to any one of claims 21 to 23, wherein the estrogen is promestriene, estriol, estradiol hemihydrate, dehydroepiandrosterone or any of their combinations.

25. Use of a kit comprising the DMSCs defined in any of claims 1 to 19 or the pharmaceutical composition defined in claim 20 or of the kit according to any of claims 21 to 24 for the treatment of chronic SUI of a subject.

26. A device comprising the DMSCs defined in any one of claims 1 to 19 or the pharmaceutical composition defined in claim 20.