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WO2021099665A1 - Cellules souches de la caduque basale du placenta à utiliser dans l'incontinence urinaire d'effort chronique - Google Patents

Cellules souches de la caduque basale du placenta à utiliser dans l'incontinence urinaire d'effort chronique Download PDF

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Publication number
WO2021099665A1
WO2021099665A1 PCT/ES2020/070718 ES2020070718W WO2021099665A1 WO 2021099665 A1 WO2021099665 A1 WO 2021099665A1 ES 2020070718 W ES2020070718 W ES 2020070718W WO 2021099665 A1 WO2021099665 A1 WO 2021099665A1
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dmscs
use according
subject
sui
cells
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Spanish (es)
Inventor
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
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Fundacion Investigacion Biomedica Hospital Universitario 12 Octubre
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Fundacion Investigacion Biomedica Hospital Universitario 12 Octubre
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Publication of WO2021099665A1 publication Critical patent/WO2021099665A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly

Definitions

  • 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.
  • Urinary incontinence is the involuntary loss of urine.
  • UI Urinary incontinence
  • SUI stress or stress UI
  • 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.
  • 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).
  • SUI cases occur after prostate surgery or radiation (Juarranz M et al. Aten. Primaria 2002 30 (5): 323-332).
  • 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).
  • drug treatment for example, alpha-adrenergic agonists, duloxetine
  • invasive treatments such as injection of fillers (eg, polytetrafluoroethylene, bovine collagen, or silicone particles) and surgery (eg, suburethral bands).
  • fillers eg, polytetrafluoroethylene, bovine collagen, or silicone particles
  • surgery eg, suburethral bands
  • UI caused by neurogenic bladder 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.
  • 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.
  • 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.
  • neurogenic incontinence has a different surgical treatment protocol.
  • Neurogenic bladder UI and SUI are different pathologies.
  • the inventors have developed a tool that allows the treatment of chronic stress urinary incontinence (SUI).
  • SUI chronic stress urinary incontinence
  • 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.
  • DMSCs injected into the suburethral mucosa reverse SUI see example 3
  • both acute SUI Figure 3
  • chronic SUI figure 4
  • 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).
  • 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.
  • DMSCs express the CD44 proteins , CD90, CD117, CD73, CD29, CD13, CD105, Oct-4, Rex1, GATA-4 and HLA class I (HLA-ABC)
  • HLA-ABC HLA class I
  • the first aspect of the invention 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.
  • DMSCs placental decidua parietalis stem cells
  • DMSCs are used as an active ingredient.
  • 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.
  • SUI is caused by deficit of the urethral sphincter.
  • 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 Mac ⁇ as 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • patients do not have a neurological pathology, for example, in the pudendal nerve, which is the cause of incontinence.
  • 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.
  • the DMSCs are administered locally in the urethra, for example, in the suburethral mucosa, intravenously, or vaginally. .
  • the DMSCs are administered through local injection into the suburethral mucosa, for example, in at least two locations.
  • 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.
  • the SUI is evaluated to decide whether the treatment has been cash or if the subject needs a new administration.
  • the DMSCs are obtained from the placenta of a mammal, for example, a human.
  • the DMSCs are obtained from human placenta.
  • 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 Mac ⁇ as MI et al. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9-495.e23.
  • 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.
  • 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.
  • the serotonin and norepinephrine reuptake inhibitor is duloxetine (eg Cymbalta®).
  • the alpha adrenergic agonist is ephedrine, pseudoephedrine, phenylpropanolamine, or any of their combinations.
  • 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.
  • promestriene for example, Colpotrofin®
  • estriol for example, Blissel®
  • estradiol hemihydrate for example, Vagifem®
  • dehydroepiandrosterone for example, Prasterone®
  • the DMSCs when used in combination with a drug, it is administered before and / or after the administration of the DMSCs.
  • 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.
  • 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.
  • 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.
  • terapéuticaally effective amount 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the serotonin and norepinephrine reuptake inhibitor is duloxetine.
  • the alpha adrenergic agonist is ephedrine, pseudoephedrine, phenylpropanolamine, or any of their combinations.
  • 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.
  • promestriene for example, Colpotrofin®
  • estriol for example, Blissel®
  • estradiol hemihydrate for example, Vagifem®
  • dehydroepiandrosterone for example, Prasterone®
  • the kit of the third aspect of the invention may contain instructions for carrying out the use of the first 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.
  • 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.
  • 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.
  • the device is a syringe.
  • the DMSCs may be marked for display.
  • 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.
  • duloxetine 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.
  • the subject presenting chronic SUI is a human.
  • the patient can be of any age, gender, or race.
  • 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.
  • 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).
  • the subject is a menopausal woman.
  • 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.
  • the subject is a woman who has previously received another treatment for SUI but has not been effective, for example , one or more surgeries.
  • 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.
  • 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.
  • 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.
  • the subject is under 65 years of age; or, alternatively, the subject is 65 years of age or older.
  • 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.
  • 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.
  • 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.
  • 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.
  • the support 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.
  • 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.
  • 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.
  • SAI suburethral mucosa of patients with SUI
  • POP pelvic
  • 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 Mac ⁇ as MI et al. 2010 Am. J. Obstet. Gynecol. 203 (5): 495.e9-495.e23. The materials and methods used are briefly indicated below:
  • 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.
  • PBS Phosphate Buffer Saline
  • 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 .
  • DMEM Modified Dulbecco Essential Medium
  • 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.
  • FISH Fluorescent in situ hybridization
  • 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.
  • permeabilization buffer (0.5% Igepal CA-630 (Sigma) diluted in 2X Sodium Citrate Saline (2X SSC)
  • reaction mixture was carried out according to the instructions indicated by the manufacturer of the Aneufast TM 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).
  • 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).
  • 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 ⁇ l of buffer.
  • 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 FITC
  • CD86 B7-2 PE
  • SSEA-1 PE SSEA-3 PE
  • SSEA-4 PE TRA-1-60 PE
  • TRA-1- 81 PE eBioscience
  • 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.
  • RT-PCR Reverse transcription and polymerase chain reaction
  • RNA ribonucleic acid
  • 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.
  • 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.
  • 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.
  • 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 Mac ⁇ as 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.
  • Cells with fibroblast morphology were obtained from the suburethral mucosa by digestion with 0.15% collagenase type II collagenase overnight at 37 ° C.
  • the cells were cultured in complete DMEM medium (Gibco) with fetal bovine serum (FBS) (Hyclon).
  • FBS fetal bovine serum
  • 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.
  • the Cell Biolabs migration kit "Cyto Select TM 24-well cell migration assay” (reference CDA-100) was used, following the manufacturer's instructions.
  • 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.
  • 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.
  • 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 1 ⁇ 2 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 TM reagent.
  • 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)
  • 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.
  • anesthesia isoflorane (AbbVie) in inhaled flow at 5% for induction of analgesia and at 2% for maintenance
  • analgesia meloxicam, 5 milligrams per milliliter, diluted 1/10 in sterile physiological saline; 200 microliters were injected, subcutaneously; Metacam®, Boehringer Ingelheim
  • the rats had a baseline measurement of their urine leakage pressures, before damage and after damage.
  • 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
  • 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).
  • 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).
  • 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.
  • DMSCs cells 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.
  • 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.
  • the test was repeated 3 times in each animal for several weeks and the mean of the LPP was calculated for each animal.
  • DMSCs cells labeled with a fluorescent marker in the Vivotrack 680nm near infrared area were used.
  • 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).

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Abstract

La présente invention concerne des cellules souches de la caduque basale du placenta à utiliser dans le traitement de l'incontinence urinaire d'effort chronique chez un sujet. La présente invention se rapporte également à la composition pharmaceutique qui comprend lesdites cellules souches de la caduque basale du placenta pour la même utilisation, ainsi que la trousse et le dispositif qui les comprend.
PCT/ES2020/070718 2019-11-22 2020-11-19 Cellules souches de la caduque basale du placenta à utiliser dans l'incontinence urinaire d'effort chronique Ceased WO2021099665A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009069991A2 (fr) * 2007-11-30 2009-06-04 Rnl Bio Co., Ltd. Agent thérapeutique cellulaire pour incontinence urinaire comprenant des cellules souches provenant de la caduque ou de l'adipose
US20180296608A1 (en) * 2015-10-02 2018-10-18 College Of Medicine Pochon Cha University Industry-Academic Cooperation Foundation Compositions containing SPHEROID CELL AGGREGATES for enhance ovary function and preparation method of the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009069991A2 (fr) * 2007-11-30 2009-06-04 Rnl Bio Co., Ltd. Agent thérapeutique cellulaire pour incontinence urinaire comprenant des cellules souches provenant de la caduque ou de l'adipose
US20180296608A1 (en) * 2015-10-02 2018-10-18 College Of Medicine Pochon Cha University Industry-Academic Cooperation Foundation Compositions containing SPHEROID CELL AGGREGATES for enhance ovary function and preparation method of the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KOIKE YUSUKE, FURUTA AKIRA, SUZUKI YASUYUKI, HONDA MARIKO, NARUOKA TAKEHITO, ASANO KOJI, EGAWA SHIN, YOSHIMURA NAOKI: "Pathophysiology of urinary incontinence in murine models", INTERNATIONAL JOURNAL OF UROLOGY : OFFICIAL JOURNAL OF THE JAPANESE UROLOGICAL ASSOCIATION AUSTRALIA, vol. 20, no. 1, 31 December 2012 (2012-12-31), pages 64 - 71, XP055825900, ISSN: 1442-2042, DOI: 10.1111/j.1442-2042.2012.03225.xpubmed:23126617 *
MACIAS M I ET AL.: "Isolation and characterization of true mesenchymal stem cells derived from human term decidua capable of multilineage differentiation into all 3 embryonic layers", AMERICAN JOURNAL OF OBSTETRICS & GYNECOLOGY, vol. 203, no. 5, 1 November 2010 (2010-11-01), pages 495.e9 - 495.e23, XP027443378, ISSN: 0002-9378, [retrieved on 20200420], DOI: 10.1016/j.ajog. 2010.06.04 5 *

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