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WO2019175459A1 - Cellules précurseurs pluripotentes obtenues à partir du muscle crémaster et leur utilisation dans la mise au point de thérapies et médecine régénérative - Google Patents

Cellules précurseurs pluripotentes obtenues à partir du muscle crémaster et leur utilisation dans la mise au point de thérapies et médecine régénérative Download PDF

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WO2019175459A1
WO2019175459A1 PCT/ES2019/070167 ES2019070167W WO2019175459A1 WO 2019175459 A1 WO2019175459 A1 WO 2019175459A1 ES 2019070167 W ES2019070167 W ES 2019070167W WO 2019175459 A1 WO2019175459 A1 WO 2019175459A1
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cells
muscle
cell
precursor cells
composition
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Inventor
Ander IZETA PERMISAN
Adolfo LÓPEZ DE MUNAIN ARREGUI
Neia NALDAIZ GASTESI
María GOICOECHEA BIANCHI
Bernardo HERRERA IMBRODA
María Fernanda LARA CABANÁS
Isabel María ARAGÓN CORTÉS
Francisco Javier MACHUCA SANTA CRUZ
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Servicio Andaluz de Salud
Administracion General de la Comunidad Autonoma de Euskadi
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Servicio Andaluz de Salud
Administracion General de la Comunidad Autonoma de Euskadi
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes

Definitions

  • Multipotent precursor cells obtained from the cremaster muscle and its use in the development of therapies and regenerative medicine
  • the present invention is within the field of regenerative medicine and cell therapy, and specifically refers to the method of isolation and expansion of multipotent precursor cells obtained from the human cremaster muscle, and its therapeutic uses.
  • Adult stem cells are characterized by their properties of self-renewal and differentiation to different lineages (multipotency) throughout the life of the individual. In general, they are responsible for the maintenance of organs and tissues in homeostasis and for their repair in response to tissue damage, be it traumatic or degenerative.
  • myogenic stem cells are known as “satellite cells,” due to their superficial position to muscle fibers, although included in the basement membrane that covers them (2).
  • the process of myogenesis is complex and involves the passage of these cells through various precursor stages (quiescence, activation, proliferation and differentiation) prior to their definitive integration through their fusion with muscle fibers within the framework of the regenerative process (3) .
  • Recognized markers of human satellite cells include Pax7, CD56, MyoD, M-cadherin and demines, but none of them are considered specific markers (4, 5).
  • the niche of satellite cells is complex: it is composed of other precursor cells, such as fibro-adipogenic precursors or FAPs, which influence the response to damage and compensatory regeneration of tissue (6).
  • the satellite cell is part of the muscular interstitial space and its niche is adjacent to capillaries and nerve endings that, in turn, influence the activation or quiescence of satellite cells by direct or paracrine signaling, as well as generating lineage precursor cells vascular or neural in response to muscle damage (7, 8).
  • CD56 + CD34 + CD144 + which is Pax7 +, accounts for 0.4% of the initial cell suspension isolated from human skeletal muscle, and which is superior in terms of its regenerative capacity compared to CD56 + myoblasts, when it is transplanted to mouse muscle ( 9).
  • CD133 + mononucleated cells isolated from human skeletal muscle although heterogeneous in nature, are capable of generating satellite cells after mouse transplantation more efficiently than myoblast cultures (10-12).
  • a Phase I clinical trial showed that muscle-isolated CD133 + cells are safe for use in humans (13), but the use of this cell population has not become widespread because there are problems in their cell expansion that must be resolved from facing a greater development of its clinical application (5).
  • ADLH + aldehyde dehydrogenase
  • the satellite cells thus defined represent approximately 2% of the cell suspension and are capable of regenerating muscle in vivo after transplantation into mouse muscle (21).
  • CD82 is a marker expressed by human satellite cells and that it can be used for its isolation, although it is not specific to them because it is still expressed in myogenic precursor cells activated or in the process of differentiation. In addition, its expression is diminished in muscular dystrophy patients, which would compromise its possible use in therapy in some types of diseases (25).
  • satellite cells still have significant problems in the face of their possible clinical application as a cell therapy drug.
  • the number of satellite cells obtained from small muscle biopsies (typically 50-100 mg) that are normally used in clinics is very limited. It is estimated that there are between 500 and 1000 satellite cells per cubic millimeter of fiber in human muscle, although their number varies between different muscle groups depending on the area and density of myofibers (23).
  • these cells expand insufficiently ex vivo or lose its stem cell characteristics as a result of in vitro culture. This has been demonstrated in both mouse (27, 28) and human (29, 30) satellite cells.
  • the isolation and characterization protocols of human satellite cells are still in development, unlike the mouse where its biology is best known (31).
  • myoblasts have been the most commonly used clinically so far.
  • the myoblasts (32) are a heterogeneous mixture of suspended cells obtained from muscle biopsies, normally used directly (without purification), or semi-purified by minimal enrichment based on the positive selection of cells that express the CD56 membrane antigen ( 33).
  • myoblasts are myogenic precursor cells, in an intermediate stage between satellite cells and terminal differentiated myocytes, although this is difficult to specify given the heterogeneity of these cultures (including those obtained from CD56 + cell fraction).
  • the cultures of myoblasts thus obtained expand in vitro with relative ease (34, 35), so they have been used in various clinical trials in indications related to skeletal and cardiac muscle, in which the results obtained have been disappointing (36- 40).
  • myoblasts can be multiple, but they are probably related to cell expansion conditions (41, 42). When culture media and conditions favor proliferation, it is easy that they also cause excessive cell differentiation, resulting in low survival, migratory capacity and fusogenicity when these cells expanded in vitro are transplanted to areas of muscle damage in vivo (5, 43).
  • Examples of signaling involved in the regulation of expansion vs. differentiation of mouse satellite cells include oxygen levels present in the culture chamber (44), changes in cell metabolism (45-47), efrins and their receptors ( 48), the FGF route (49), the Notch route (50), SOX7 (51), TAZ / YA P (52) and the TGFbeta / Smad route (53), among others. Even so, it is important to note that the vast majority of studies in this regard are carried out in animal models and their translation to human cells is uncertain (54).
  • myoblast cultures retain a minor cell subpopulation (slower cell cycle), which is also responsible for taking the transplant once the cultures are injected into the recipient animal, while the rest of the cells enter apoptosis in the first hours after the transplant (55). Pre-selection of these slow-cycle cells prior to transplantation greatly improved the results thereof (56).
  • human myoblast cultures With respect to human myoblast cultures, it is known that these heterogeneous cultures are capable of regenerating muscle (57) and even generating human satellite cells after transplantation into mice (58). Laumonier and cois propose that human myoblast cultures retain a population of “reserve” satellite cells, identified as CD45-CD34-CD144-CD56 + CD146 + after 5-7 days of culture (59). These cells are Pax7 + MyoD-Myf5 + (that is, similar to quiescent satellite cells) and show better survival than myoblasts after mouse transplantation.
  • myoblast cultures may be successful in repairing small muscles such as those affected in the oculopharyngeal muscular dystrophy.
  • very large amounts of cells are being injected in the ongoing clinical trials (hundreds of millions; (60)), indicating that the regeneration potential of these cultures is clearly improved and that it would be desirable to obtain purer cell populations to reduce the expansion of cell therapy products (to avoid the risks associated with the long periods of expansion required).
  • co-injection of other precursor cells that facilitate regeneration, revascularization and reinnervation of the damaged area could also be useful.
  • mouse and rat muscle derived cells in three-dimensional culture in the form of floating myospheres enriches them in Pax7 + MyoD + cells, potentially muscle satellite cells, which differentiated in vitro to mononucleated contractile cells and contributed to the regeneration of muscle fibers after transplantation to mouse muscle (63).
  • mouse myospheres contain at least two cell subpopulations, a myogenic precursor cell (possibly satellite cell) that is ITGA7 + Myf5 + MyoD + Pax7 + and another non-myogenic precursor cell (possibly the fibroadipogenic precursor-FAP) characterized as PDGFRa + Sca-1 + (64, 65). It is important to note that both precursor cells are key to the proper functioning of the muscle, both in homeostasis and in regeneration, so that the culture of myospheres would have the added advantage of providing at least two types of precursor cells, instead of one.
  • the culture of myospheres also contains a third type of precursor cells, the neural precursors, as we have previously shown in the characterization of myogenic spheres isolated from the mouse dermis (66).
  • the biopsies were a minimum of 100 mg to establish the spherogenic culture, and could be expanded for 20 weeks (18 passes), although the cultures began to grow slower after week 16.
  • These precursor cells possibly satellite cells are defined as CD34-CD45-Pax7 + CD56 + ALDH1 + and, surprisingly, they are also defined as Oct3 / 4 + Nanog + Sox2 +, indicating their pluripotency status in these growing conditions. Only 50% of these cells expressed demining and MyoD in early passes, demonstrating the heterogeneity of the subpopulation under study. An additional cause of concern with this study is the possible contamination with cancer cells.
  • Muscle regeneration by means of cell therapy protocols based on myogenic, vasculogenic precursor cells that promote reinnervation can be applied in various skeletal and cardiac muscle dysfunctions, such as congenital myopathies (69), muscular dystrophies and other neuromuscular degenerative diseases (70 ), cardiac dysfunction (71), loss of volumetric muscle mass, cachexia or sarcopenia (72).
  • satellite cells could present divergent identities based on their axial / anatomical location or the type of muscle fiber they come from (3).
  • the Proxl transcriptional repressor is characteristic of slow muscle fibers and is expressed in satellite cells (77).
  • myogenic cells have been isolated from diverse muscles and not necessarily identical or close (from an anatomical or developmental biology point of view) to those of the intended area of application (5).
  • the use of precursor cells obtained from regions similar to the target regions may have associated advantages in terms of functionality, adaptability or survival thereof.
  • Some similar examples in this regard may be (a) obtaining CD56 + myoblasts from the pyramidal muscle in patients who will undergo radical prostatectomy (78) and (b) extracting myoblasts from the abdominal rectus muscle (79), so that they can be used later in cases of urinary incontinence.
  • Urinary incontinence (UI) is a prevalent pathology, which affects more than 200 million women and 17% of men worldwide; although your diagnosis may be underestimated.
  • Data in animal models suggest that injection of precursor cells may improve weakened sphincter function (75). In fact, clinical trials that have used autologous injection of muscle precursor cells obtained from muscle biopsies demonstrate that this therapy is safe and has acceptable results.
  • the authors of the present invention propose that the use of multipotent precursor cells (myogenic, vascular and neural) isolated from the cremaster muscle presents advantages for cell therapy in patients of urinary or anal incontinence with sphincter damage or in the treatment of muscle groups of small size such as oculopharyngeal muscular dystrophy or facioscapulohumeral dystrophy, in a single treatment or in combination with other active substances, or associated with a medical device.
  • the cremaster is a striated muscle (although it lacks tendon insertion) and voluntary control that is intermingled with abundant smooth muscle fibers. It contains predominantly type 1 (slow) fibers, but also some type II B (very fast), and whose function in the adult is to contribute to thermoregulation and the protection of the testicles as one of the tunics that form the scrotum. Thanks to the crematoric reflex, its electrophysiological properties are well known. The muscle is densely innervated and has numerous motor plates, which explains its abundant spontaneous discharges.
  • FIG. 1 Characterization of the male crema muscle.
  • A Anatomical location of the cremaster muscle. The biopsy collection area is framed.
  • B Histological section with hematoxylin-eosin staining where striated and smooth muscle fiber bundles are observed (b ’, detail at higher magnification).
  • CD By immunofluorescence, it is observed that myosin positive striated fibers (MyHC +, green) are surrounded by basement membrane (Laminin +, red). The cores are shown in blue.
  • HEY Analysis of the composition of myosin in type I fibers (slow, green) and II (fast, green). The laminin is shown in red and the nuclei in blue. I-J.
  • FIG. 1 Multipotent precursor cells present in mouse dermospheres.
  • Figure 3. Detection of myogenic precursor cells of the cremester muscle in myospheres.
  • the arrow in panel B indicates undissociated remains of muscle.
  • DF Analysis of the expression of myogenic markers by immunofluorescence. Staining (in green) was detected for the Pax7 (D), MyoD1 (E) and MyHC (F) markers. The cores are shown in blue.
  • Figure 4 Detection of neurogenic and vasculogenic precursor cells of the cremaster muscle in myospheres.
  • A-E Analysis of the expression of neurogenic and vasculogenic precursor markers by immunofluorescence. Staining was detected in green for marker CD56 (A), in red for p75NTR (B) and in blue (C) for nuclei (DAPI).
  • Panel D shows overlapping A-B panels, and E overlays A-B-C. The following symbols show neurogenic cells (p75NTR + CD56 +, asterisk), vasculogenic cells (p75NTR + CD56-, arrow) and p75NTR-CD56 + cells (arrowhead).
  • FIG. 5 Differentiation in vitro of the precursor cells of the cremaster under myogenic conditions.
  • A Temporary diagram of myotube formation from disintegrated spheres.
  • B Contrast image of phases of the culture of spheres disintegrated at day 0 in differentiation medium.
  • C Phase contrast image of multinucleated myotubes formed after 7 days in culture.
  • D-G Characterization of myotubes by immunofluorescence with anti-MyHC antibodies (green) and Pax7 (green). The cores are shown in blue.
  • H Analysis of gene expression determinants for myogenesis (Pax7, Myf5, MyoD1, Myogenin, MyHC3 and MyHC2) by retrotranscription followed by quantitative real-time PCR (RT-qPCR). The results obtained in relation to the Tbp control gene are shown.
  • FIG. 6 Regenerative capacity in vivo of the precursor cells of the cremaster.
  • the authors of the present invention propose that the use of multipotent precursor cells (myogenic, vascular and neural) isolated from the cremaster muscle presents advantages for cell therapy in patients of urinary or anal incontinence with sphincter damage or in the treatment of muscle groups of small size such as oculopharyngeal muscular dystrophy or facioscapulohumeral dystrophy, in a single treatment or in combination with other active substances, or associated with a medical device.
  • the cremaster muscle has not been used as a source of precursor cells.
  • the main function of the cremaster is the descent of the testis to the inguinal ring through its contraction, so obtaining a biopsy would not involve adult patients with significant functional damage in that area and could be used as a source of multipotent cells for the study and treatment of patients with traumatic or degenerative disorders that affect muscle groups.
  • the cremaster muscle represents a valuable source for isolating multipotent precursor cells capable of structurally and biochemically replacing: i) the muscular wall of the urinary sphincter, anal, or other organs or tissues that include striated muscle fibers or bundles, ii) replacement of damaged or afunctional urinary or anal sphincter myogenic cells or damaged striated muscle in this or other muscle pathologies, iii) recovery of striated muscle, urinary sphincter, or other organs, such as the anal sphincter, or it can also be applied in various skeletal and cardiac muscle dysfunctions, such as congenital myopathies, muscular dystrophies and other neuromuscular degenerative diseases, cardiac dysfunction, loss of volumetric muscle mass, cachexia or sarcopenia, iv) replacement of useful myogenic cells hoisted as a constituent part of organs generated by tissue engineering techniques such as the urethra, trachea and other tissues and organs,
  • the authors of the present invention are the first who have developed a method to obtain multipotent precursor cells isolated from the cremaster muscle.
  • a first aspect of the invention relates to a composition
  • a composition comprising a cell population of multipotent precursor cells derived or derived from human cremaster muscle, hereinafter referred to as a composition of multipotent precursor cells of the invention, wherein said population, at its instead, it represents a heterogeneous population of cells that includes various types of precursor cells, as illustrated in Figure 2, such as myogenic precursor cells, vasculogenic precursor cells, and neurogenic precursor cells.
  • the referred cell population hereafter referred to as a cell population of multipotent precursor cells, must comprise at least one multipotent precursor cell derived from or derived from the human cremaster muscle.
  • said cell population comprises at least 20%, preferably 30%, more preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% of multipotent precursor cells derived or originating from human cremaster muscle.
  • biopsies are started of the cremaster muscle, said tissue biopsy is chopped into small fragments and, once the tissue has been broken down, the multipotent precursor cells of the cremaster muscle are obtained, and then cultured (see examples of the invention).
  • a second aspect of the invention relates to a composition comprising myospheres obtained from cultures of a cell population of cells isolated from the cremaster muscle of the first aspect of the invention.
  • the composition obtained from the first aspect of the invention is re-suspended in culture medium for non-adherent conditions, called proliferation medium (Neurobasal TM A medium supplemented with 2% of supplement B27 50X, 1% of 200 mM L-glutamine and 1% of solution of penicillin / streptomycin 100X) and seeded in untreated plates to obtain a culture of myospheres.
  • proliferation medium Neurosporasal TM A medium supplemented with 2% of supplement B27 50X, 1% of 200 mM L-glutamine and 1% of solution of penicillin / streptomycin 100X
  • the cell precipitate resulting from cell isolation is resuspended in 1 ml_ of suspension culture medium and cell viability is counted and monitored before being transferred to a well of the untreated 6-well plates, where more medium is added for suspension culture up to 5 ml_. If the viable cell count turns out to be less than 500,000 cells / mL, they are transferred to a smaller well of untreated 12-well plates, and the medium and factor volumes are adjusted in proportion to what is established. These plates prevent the adhesion of the cells and favor the formation of cell spheres.
  • LSGS Low serum growth supplement
  • rrFGF2 Rat recombinant basic fibroblast growth factor
  • rrEGF Rat recombinant epidermal growth factor
  • the expression of myosin isoforms (labeled by MyHC) in precursor cells in a mononuclear state is also indicative of the myogenic precursor state of the same. Therefore, in a preferred embodiment of the first or second aspect of the invention, the composition comprising multipotent precursor cells isolated from the human cremaster muscle of the first aspect of the invention or the composition comprising spheres obtained from cultures of cells isolated from the cremaster muscle of the first aspect of the invention, it is characterized in that said cell population is positive for Pax7, MyoD1 and MyHC.
  • the p75NTR and CD56 markers are observed in the fields obtained from cultures of isolated cells of the cremater muscle of the first aspect of the invention ( Figure 4).
  • the composition comprising multipotent precursor cells isolated from the human cremaster muscle of the first aspect of the invention as well as the composition comprising spheres obtained from cultures of cells isolated from the cremaster muscle of the first aspect of the invention, is characterized by comprising said assembly.
  • composition of the first aspect of the invention as well as the composition of the second aspect of the invention are especially useful for carrying out transplants in damaged muscles.
  • marker is understood as a protein that distinguishes a cell (or group of cells) from another cell (or group of cells).
  • a protein that is expressed on the surface of myogenic cells but not in other cells of a cell population acts as a marker protein for myogenic precursor cells.
  • “Positive” means that the cell naturally expresses the marker. To consider that the marker is expressed, it must be present at a “detectable level”. In this report, “detectable level” means that the marker can be detected by one of the standard methodologies, such as PCR, blotting, immunofluorescence or FACS.
  • a gene is expressed by a cell of the invention if it can be reasonably detected after 20 cycles, preferably 25 cycles, and more preferably 30 cycles of PCR, corresponding to a level of expression in the cell of at least 100 copies per cell. It is considered that a marker is not expressed by a cell of the invention, if the expression cannot be detected at a level of about 10-20 copies per cell. Among these positive / negative levels, the cell may be weakly positive for a given marker.
  • the cell population of the invention is positive for a certain marker if at least 20% of the cells in the population show a detectable expression of the marker, preferably 70%, 80%, 90%, 95% , and much more preferably, 98%. Sometimes, 99% or 100% of the cells of the invention show a detectable expression of the marker.
  • expression can be detected, for example but not limited, by PCR techniques, using FACS (fluorescence activated cell sorting), or by immunohistochemistry using specific antibodies.
  • naturally expressed means that the cells have not been manipulated by recombinant technology, in any way, that is, for example, that the cells have not been artificially induced to express these markers or modulate the expression. of these markers by the introduction into the cells of exogenous material, such as the introduction of heterologous promoters, or other sequences operatively linked to any of the endogenous genes, or by the introduction of exogenous genes.
  • the population of myogenic precursor cells present in the composition of the first or second aspect of the invention derives from the cremater's satellite cells and maintains the stem cell capabilities thanks to its self-renewal in the Myosphere culture described in the second aspect of the invention, equivalent to what is known as "reserve satellite cell” in traditional myoblast cultures.
  • the "satellite of the cremaster" cell is understood as the stem cell from the cremaster muscle, which has the ability to regenerate muscle throughout the life of the individual and differentiate into myogenic precursor cells.
  • the obtaining of myogenic cells was carried out from the formation of myospheres present in the proliferation culture of the second aspect of the invention.
  • the culture conditions to obtain a myogenic differentiation ( Figure 5).
  • the myospheres were disintegrated with 0.25% Trypsin-EDTA for 5 minutes at 37 ° C and then seeded the precursor cells on a natural extracellular matrix (ECM) that favors cell adhesion and differentiation (66, 88), composed of extract of basement membrane Cultrex® (2.77 mg / mL in final concentration), Netrin-4 (0.83 pg / mL), Netrin-G1a (0.83 pg / mL) and low molecular weight hyaluronic acid (2, 5 mg / mL) in PBS (pH 7.4).
  • ECM extracellular matrix
  • the extracellular matrix solution was prepared taking into account the concentration of the Cultrex® basement membrane extract (Cultrex), depending on the product batch. Based on this final volume data, the volumes of the rest of the compounds were adjusted. In all cases an extracellular matrix solution was prepared at a 1: 3 dilution, first preparing a mixture to dissolve the reagents (equivalent to 1 of the 1: 3 dilution) and then adding the necessary volume of PBS (pH 7.4 ) as diluent to the mixture to obtain the final solution (equivalent to 2 resulting from the calculation of the 1: 3 dilution).
  • Cultrex® basement membrane extract Cultrex
  • an extracellular matrix was prepared with all the mentioned reagents or an extracellular matrix without the presence of Netrina-4 and Netrina-G1a, adjusting in this last case the volume of PBS (pH 7.4) that would be used first to dissolve and mix the compounds. Since the compounds used are thermosensitive, in order to maintain their integrity and avoid premature polymerization, all reagents were gradually thawed and kept on ice throughout the process together with the extracellular matrix solution in preparation. So that after reconstituting and thawing all the compounds, the mixing solution was prepared in a 50 mL Falcon® which was kept on ice, and then PBS (pH 7.4) was added as diluent.
  • the freshly prepared extracellular matrix was deposited in the form of a drop (300 ml / well) in sterile 4 or 24 well plates in which autoclaved coverslips had previously been placed and spread by tilting the plate to form a uniform film. Once the matrix was deposited, the plates were kept for 24 hours at 37 ° C. After this time, the excess liquid accumulated in the wells was removed and allowed to dry in the laminar flow hood for 15 minutes in order to obtain the desired substrate.
  • the pellet aggregated spheres were resuspended in 1 mL of 0.25% Trypsin-EDTA solution and disintegrated keeping them 5-7 minutes at 37 ° C to obtain a suspension of single cells in order to be able to count them, deposit them separately and achieve a more homogeneous differentiation.
  • the Enzymatic action was stopped by diluting the solution with PBS (pH 7.2) and removing it after a 5 minute centrifugation at 1500 rpm and then resuspend the cell pellet in 1 ml_ of medium for adhesion culture and count the number of viable cells.
  • a third aspect of the invention relates to a composition
  • a composition comprising a cellular population of myogenic cells derived from the formation of the myospheres present in the proliferation culture of the second aspect of the invention or of a cellular composition comprising a cell population of multipotent precursor cells derived or from human crema muscle.
  • said cellular composition of myogenic cells is characterized in that it comprises a positive cell population for the myogenic genes Pax7, Myf5, MyoD1, Myogenin, MyHC3 and MyHC2, in greater amount compared to a control negative (such as biopsies taken from the same region but not muscular).
  • the referred cell population hereafter referred to as the myogenic cell cell population, must comprise at least one myogenic cell derived or from the human cremaster muscle.
  • the cell population comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% of myogenic cells derived from or coming from the Human cremaster muscle.
  • vasculogenic or neurogenic precursor cells can also be obtained from the formation of the myospheres present in the proliferation culture of the second aspect of the invention or from a cellular composition comprising multipotent precursor cells derived or originating from the cremaster muscle human.
  • a fourth aspect of the invention relates to a composition comprising a cell population of vasculogenic precursor cells derived from the formation of the myospheres present in the proliferation culture of the second aspect of the invention or of a cellular composition comprising a cell population of multipotent precursor cells derived or derived from human cremaster muscle.
  • Said cellular composition is characterized in that it comprises a positive cell population for: p75NTR, SOX2, PAX3, DLK1, RGS5, AOC3, ITGA7, ECRG4 and negative for the CD56 marker ( Figure 4).
  • the referred cell population henceforth the cell population of vasculogenic precursor cells, must comprise at least one vasculogenic precursor cell derived from or derived from the human cremaster muscle.
  • the cell population comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% of vasculogenic precursor cells derived or from of the human cremaster muscle.
  • a fifth aspect of the invention relates to a composition
  • a composition comprising a cellular population of neurogenic precursor cells derived from the formation of the myospheres present in the proliferation culture of the second aspect of the invention or of a cellular composition comprising a population cell of multipotent precursor cells derived or from human crema muscle.
  • Said cell composition is characterized in that it comprises a positive cell population for: p75NTR, SOX2, PAX3, CD56, SOX10, ERBB3, L1CAM, CDH2, CDH19, PMP22, PLP1, DHH and negative for the SOX9 marker (Figure 4).
  • the aforementioned cell population henceforth the cellular population of neurogenic precursor cells, must comprise at least one neurogenic precursor cell derived from or derived from the human cremaster muscle.
  • the cell population comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% of derived or derived neurogenic precursor cells. of the human cremaster muscle.
  • the cells present in any of the compositions of the invention can be genetically modified by any conventional method including, by way of illustration, not limitation, transgenesis processes, deletions or insertions in their genome that modify the expression of genes that are important for its basic properties (proliferation, migration, differentiation, etc.), or by inserting nucleotide sequences that encode proteins of interest such as, by example, proteins with therapeutic properties. Therefore, in another preferred embodiment, the cells present in any of the compositions of the invention have been genetically modified.
  • the cells present in any of the compositions of the invention can be clonally expanded using a method suitable for cloning cell populations.
  • a proliferated population of cells can be physically collected and seeded on a separate plate (or the wells of a "multi-well” plate).
  • cells can be subcloned into a "multi-well” plate in a statistical relationship to facilitate the operation of placing a single cell in each well (for example, from about 0.1 to about one cell / well or even from about 0.25 to 0.5 cells / well, such as 0.5 cells / well).
  • the cells can be cloned at low density (for example, in a Petri dish or other suitable substrate) and isolated from other cells using devices such as cloning rings.
  • the production of a clonal population can be expanded in any suitable culture medium.
  • isolated cells can be cultured to a suitable point when their development phenotype can be evaluated.
  • the cell population of any aspect of the invention is an isolated population.
  • isolated indicates that the cell or the cell population of the invention to which it refers, are not in their natural environment. That is, the cell or cell population has been separated from its surrounding tissue. Particularly it means that said cell or cell population is substantially free (free) of other cells normally present in the tissue, or derived therefrom, from a subject from which the muscle has been removed for the isolation of said cell or cell population. .
  • a cell is essentially free of other cells present in the environment thereof when separated from at least 60%, preferably at least 80%, preferably at least 90%, more preferably of at least 95%, even more preferably of at least 96%, 97%, 98% or even 99%, of other cells normally present in said environment.
  • the composition of the second aspect of the invention is obtained by a method, hereafter referred to as a method of obtaining multipotent precursor cells of the invention, which comprises a) disintegrating biopsied tissue from the cremaster muscle, b) separate the intact (viable) cells from the cell debris c) cultivate the cells from step (b) in suspension until the formation of spheres (5-8 days).
  • the disintegration of step (a) can be carried out by different methods, including mechanical methods, enzymatic methods, or a combination of both.
  • the enzymes used are: acutase, collagenase (of various types, such as A, I, II, IV, or XI; or mixtures thereof), dispase, DNase I, elastase, hyaluronidase, liberase, papain, pronase, trypsin -EDTA, TrypLE, and very often combinations of the above.
  • acutase collagenase (of various types, such as A, I, II, IV, or XI; or mixtures thereof), dispase, DNase I, elastase, hyaluronidase, liberase, papain, pronase, trypsin -EDTA, TrypLE, and very often combinations of the above.
  • type I collagenase or commercial mixtures of different collagenases such as libera
  • the separation of step (b) can be carried out by different methods, among them, but not limited to: filtration, decantation, centrifugation, magnetic or FACS-based separation methods, and so on.
  • Step (c) is performed in a proliferation medium that does not contain serum and under conditions of non-adherence to the substrate.
  • This medium at least contains: a "base" culture medium such as Neurobasal and precursor cell growth factors that may include LSGS, FGF-2 and EGF, among others.
  • the days of cultivation between pass and pass vary between 4 and 8 days, depending on various factors, and preferably at least 7 days to obtain spheres of adequate size for later manipulation.
  • the "base” culture medium may be, preferably but not limited to, a medium designed for the maintenance and long-term maturation of populations of pure prenatal and embryonic neuronal cells, such as the Neurobasal medium. More preferably the basal medium is supplemented with B27, and even more preferably with L-glutamine and an antibiotic solution. Between step (b) and (c) there may preferably be a centrifugation step to obtain a cell precipitate.
  • the spheres of the composition of the second aspect of the invention can be selected and disintegrated to be injected directly, for example for the treatment of urinary incontinence, they can be used directly in therapy in the form of myospheres (without cell disintegration) or they can be differentiated from myoblasts or striated muscle cells to use muscle constructs as therapeutic agents or for their usefulness in the screening of molecules, biologicals or other therapeutic agents whose target is skeletal muscle. In the latter cases it would be preferable to use the cells with support media, such as a matrix.
  • the method of obtaining multipotent precursor cells of the invention further comprises: d) differentiating multipotent precursor cells from the spheres of step c )
  • Precursor cells can be differentiated based on various protocols existing in the literature, or identically to how myoblast cultures differ.
  • the differentiation of step d) is to myoblasts.
  • step d) is to myotubes.
  • multipotent precursor cells of the cremaster it is preferable to use multipotent precursor cells of the cremaster than satellite cells, since the satellite cells are difficult to grow and the number of cells obtained is small, and in addition these cultures do not have vasculogenic or neurogenic capacity.
  • the myogenic precursor cells of the cremaster have in turn the ability to proliferate and give rise to myoblasts, or to remain as precursor cells in culture thanks to their capacity for self-renewal.
  • a seventh aspect of the invention relates to a composition as defined in any of the aspects or embodiments outlined in above, wherein said composition is a pharmaceutical composition (hereinafter "the pharmaceutical composition of the present invention").
  • the pharmaceutical composition of the present invention relates to the compositions of any of the first, second or third aspects of the invention.
  • the pharmaceutical composition of the present invention comprises a combination of any of the compositions of the invention. Even more preferably the pharmaceutical composition comprises a combination of the composition of the third, fourth and fifth aspects of the invention.
  • the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.
  • compositions of the present invention can be used in a treatment method in isolation or together with other pharmaceutical compounds. Therefore, in another more preferred embodiment of this aspect of the invention, the pharmaceutical composition of the present invention further comprises another active ingredient.
  • active component means any component that potentially provides a pharmacological activity or other different effect on diagnosis, cure , the mitigation, treatment, or prevention of a disease, or that affects the structure or function of the human body or other animals.
  • active components of biological origin include growth factors, hormones and cytokines.
  • a variety of therapeutic agents are known in the state of the art and could be identified by their effects. Certain therapeutic agents are able to regulate cell proliferation and differentiation.
  • nucleotides examples include nucleotides, chemotherapeutic drugs, hormones, non-specific proteins (other than antibodies), oligonucleotides (for example, anti-sense oligonucleotides that bind to a target nucleid acid sequence (e.g., RNA sequence), peptides and Peptidomimetics: Other cells may also act as active components.
  • oligonucleotides for example, anti-sense oligonucleotides that bind to a target nucleid acid sequence (e.g., RNA sequence), peptides and Peptidomimetics:
  • Other cells may also act as active components.
  • the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipient refers to the fact that it must be approved by a regulatory agency of a federal government or a national government or one listed in the United States Pharmacopoeia or European Pharmacopoeia, or some other pharmacopoeia generally recognized for use in animals and humans.
  • carrier refers to a diluent, excipient, carrier or adjuvant with which the stem cells, progenitor cells or differentiated cells of the invention, the cells of the invention, as well as the cells of the cell population of the invention , must be administered; obviously, said vehicle must be compatible with the cells.
  • said vehicle include any physiologically compatible vehicle, for example, isotonic solutions (for example, sterile saline (0.9% NaCI), phosphate buffered saline (PBS), Ringer-lactate solution, etc.), optionally supplemented with serum, preferably with autologous serum; culture media (eg, DMEM, RPMI, McCoy, etc.), or, preferably, a solid, semi-solid, gelatinous or viscous support medium, such as collagen, collagen-glycosamino-glycan, fibrin, polyvinyl chloride, poly acids -amino acids, such as polylysine, or polyiorithin, hydrogels, agarose, silicone dextran sulfate.
  • physiologically compatible vehicle for example, isotonic solutions (for example, sterile saline (0.9% NaCI), phosphate buffered saline (PBS), Ringer-lactate solution, etc.), optionally supplemented with serum, preferably with auto
  • the support medium may in special embodiments, contain, growth factors or other agents.
  • the cells can be introduced into the liquid phase of a vehicle that is subsequently treated so that it becomes a more solid phase.
  • said vehicle in which the vehicle has a solid structure, said vehicle can be configured according to the shape of the lesion, such as in the form of a ring for the treatment of urinary incontinence due to muscle damage striatum of the external urinary sphincter.
  • the pharmaceutical composition of the invention may, if desired, also contain, when necessary, additives to increase and / or control the desired therapeutic effect of the cells, for example buffering agents, active surface agents, preservatives, etc.
  • the pharmaceutically acceptable carrier may comprise a cell culture medium that maintains the viability of the cells.
  • the medium will generally be serum free in order to avoid causing an immune response in the recipient.
  • the carrier will generally be buffer and / or pyrogen free.
  • metal chelating agents for the stabilization of the cell suspension, it is possible to add metal chelating agents.
  • the stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by the addition of additional substances, such as, for example, aspartic acid, glutamic acid, etc.
  • Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to one skilled in the art and are normally used in the production of cellular compositions.
  • suitable pharmaceutical vehicles are described in "Flemington's Pharmaceutical Sciences” by EW Martin. Additional information on these vehicles can be found in any manual of pharmaceutical technology (ie Galenic pharmacy).
  • the pharmaceutical composition of the invention can be administered in a suitable pharmaceutical form of administration. Therefore, the pharmaceutical composition of the invention will be formulated according to the chosen form of administration. The formulation will adapt to the form of administration.
  • the pharmaceutical composition is prepared in a liquid, solid or semi-solid dosage form, for example, in the form of a suspension, in order to be administered by implantation, injection or infusion to the subject in need of treatment.
  • a possible formulation of the pharmaceutical composition of the invention is presented in a sterile suspension with a pharmaceutically acceptable excipient, for example, an isotonic solution, for example, phosphate buffered saline (PBS), or any other suitable pharmaceutically acceptable vehicle for administration to a subject parenterally, although other routes of administration may also be used.
  • a pharmaceutically acceptable excipient for example, an isotonic solution, for example, phosphate buffered saline (PBS), or any other suitable pharmaceutically acceptable vehicle for administration to a subject parenterally, although other routes of administration may also be used.
  • PBS phosphate buffered saline
  • the administration of the pharmaceutical composition of the invention to the subject in need will be carried out using conventional means.
  • the pharmaceutical composition of the invention can be administered to the subject parenterally by suitable devices, such as syringes, catheters, trocars, cannulas, etc.
  • suitable devices such as syringes, catheters, trocars, cannulas, etc.
  • the pharmaceutical composition of the invention can be administered using equipment, apparatus and devices suitable for the administration of cellular compositions and known to a person skilled in the art.
  • direct administration of the pharmaceutical composition of the invention to the site that is intended to benefit could be advantageous.
  • the direct administration of the pharmaceutical composition of the invention for the desired organ or tissue can be achieved by direct administration (for example, by injection, etc.) on the external surface of the organ or tissue affected by the insertion of a suitable device, for example, a suitable cannula, by infusion (including reverse flow mechanisms) or by other means described in this patent or known in the state of the art.
  • a suitable device for example, a suitable cannula
  • infusion including reverse flow mechanisms
  • the pharmaceutical composition of the invention can be stored until its application by conventional methods known to those skilled in the art.
  • the pharmaceutical composition of the invention can be stored at room temperature or lower, in a sealed container, supplemented or not with a nutrient solution.
  • medium-term storage (less than 48 hours) it is preferably carried out between 2-8 ° C, and in the pharmaceutical composition of the invention a buffered isosmotic solution is also included in a container that is made or coated with a material that prevents cell adhesion.
  • Longer term storage is preferably carried out by cryopreservation and stored under conditions that promote the conservation of cellular function.
  • the pharmaceutical composition of the invention can be used in combination therapy.
  • additional drugs could be part of the same pharmaceutical composition or, alternatively, be supplied in the form of a separate composition for simultaneous or successive administration (sequential in time) to the administration of the pharmaceutical composition of the invention.
  • compositions of the invention can be implanted or injected into the patient together with a supporting material component. This could ensure that the cells remain in the right place within the patient.
  • the support material is of natural or synthetic origin.
  • the support material of natural origin is selected from the list consisting of: silk, decellularized bovine mesenteric serous membranes, decellularized bovine pericardium and combinations thereof.
  • the support material is thread with a monofilament or multifilament structure, and in a particular embodiment, the support material is a silk nanofiber sheet.
  • support material examples include collagen, fibrin, laminin, fibronectin and an artificial material, or combinations thereof, as a basis. This list is provided by way of illustration only, and is not intended to be limiting. It would be obvious to the person skilled in the art to use any combination of one or more components to form the matrix.
  • the cells of the compositions of the invention may be contained within a microsphere. Within this embodiment, the cells can be encapsulated within the center of the microsphere. Also within this embodiment, the cells may be embedded in the microsphere matrix material.
  • the matrix material may include any suitable biodegradable polymer, including but not limited to, alginates, polyethylene glycol (PLGA), fibrin and sericin and polyurethanes, or any combination thereof. This list is provided by way of example only, and is not intended to be limiting.
  • the cells of the compositions of the invention may adhere to a medical device, hereinafter medical device of the invention, intended for implantation.
  • medical devices include stents, pins, stitches, fractures, pacemakers, joint prostheses, artificial skin, and rods.
  • This list is provided by way of illustration only, and is not intended to be limiting. It would be obvious to the person skilled in the art to adhere the cells to the medical device by different methods.
  • cells can adhere to the medical device using fibrin, one or more members of the integrin family, one or more members of the cadherin family, one or more members of the selectin family, one or more molecules cell adhesion (CAMs), one or more members of the immunoglobulin family and one or more artificial adherents.
  • CAMs molecules cell adhesion
  • an eighth aspect of the invention relates to a medical device, hereafter referred to as a medical device of the invention, comprising the cells of the compositions of the invention.
  • a ninth aspect of the invention relates to an in vitro method for the preparation of an artificial tissue, hereinafter artificial tissue of the invention, comprising: a) sowing in a support material the cells of any of the compositions of the invention, and b) culturing said cells in the support material of (a) in an appropriate culture medium.
  • Another aspect of the invention relates to the artificial tissue of the invention obtained by the in vitro method described above.
  • That the culture medium is suitable for obtaining artificial tissue is known in the state of the art.
  • the culture medium in the case of cells derived from the muscle mass that constitute a total population of cells, it should contain, for example, Neurobasal A medium, supplements (B27 and L-Glutamine) and growth factors such as FGF, EGF, LSGS.
  • “Support” as used herein, refers to any device or material that can serve as a base or matrix for the growth of the cells derived from the cremaster muscle that constitute a total population of multipotent precursor cells of the invention, and more preferably for the growth and differentiation of the multipotent precursor cells of the invention.
  • the support material is of natural or synthetic origin.
  • the support material of natural origin is selected from the list consisting of: silk, decellularized bovine serous membranes isolated from the mesentery, decellularized bovine pericardium and combinations thereof.
  • the support material is thread with a monofilament or multifilament structure, and in a particular embodiment, the support material is a silk nanofiber sheet.
  • support material examples include collagen, fibrin, laminin, fibronectin, one or more polysaccharides, and an artificial material, or any combination thereof. This list is provided by way of illustration only, and is not intended to be limiting. It would be obvious to the person skilled in the art to use any biomaterial, conventional or advanced orthopedic biomaterial or a combination of biomaterials.
  • the support material is thread with monofilament or multifilament structure.
  • tissue suture One of the major drawbacks of tissue suture is the fact that the diameter of the needle is larger than the thread, so that the point of the insertion needle will not be fully occupied by the latter, the generation of the zones through which fluid loss can occur.
  • This poor wound closure is often associated with postoperative complications, such as intestinal anastomosis performed in patients with carcinoma or diverticulosis, which are performed due to resection for an intestinal disease, subsequently joining the two healthy ends.
  • feces could be lost and invading surrounding tissues, which it would cause peritonitis, with the consequent risk to the patient's life.
  • This risk is increased in patients with a reduced thickness of the intestinal wall, as in the case of an inflammatory bowel disease.
  • Multipotent precursor cells can be applied in the suture so that the opening is sealed generated by the passage of the thread through the seam. Therefore, in another preferred embodiment, the support material is a suture attached to a needle.
  • suture refers to a thread or fiber or other closure material that can be used to sew a wound.
  • staples are an alternative to the classic suture method. It allows the primary closure of tissue in less time, reduce blood loss, reduce contamination and preserve blood flow.
  • a limiting factor in the use of staples as a primary care healing method is the possibility of having access to the upper and lower part of the tissue to be joined. Also, due to the force exerted the insertion of the staples can cause tearing of the tissue. Then, in another preferred embodiment, the clips are the support material.
  • Another aspect of the invention relates to the use of a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, or c) the artificial tissue of the In the preparation of a medicament, or alternatively, it refers to a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, c) artificial tissue of the invention, for use in medicine or therapy.
  • Another aspect of the invention relates to the use of a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, c) the artificial tissue of the invention, in the preparation of a medicament for increasing partially or completely, restore or replace the functional activity of a diseased or damaged tissue or organ, or alternatively, refers to a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, c) the artificial tissue of the invention, to partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ.
  • the diseased or damaged tissue or organ is the urethral sphincter, the anal sphincter, or a combination of both.
  • another aspect of the invention relates to the use of a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, c) the tissue of the invention, in the preparation of a medicament for the treatment of urinary incontinence, fecal incontinence, or both, or alternatively, refers to a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, c) the artificial tissue of the invention, for the treatment of urinary incontinence, fecal incontinence, or both.
  • Another aspect of the invention relates to the use of a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, c) the artificial tissue of the invention, in the preparation of a medicament for the regeneration of striated muscle, or alternatively, refers to a) any of the compositions of the invention, preferably the composition of the first, second or third aspect of the invention, b) the medical device of the invention, c) the artificial tissue of the invention, for the regeneration of striated muscle.
  • Figure 2 shows the spheres obtained from cell cultures obtained from mouse dermis, and how those dermospheres contain multipotent precursor cells of various types, including myogenic precursor cells, vasculogenic precursor cells and neurogenic precursor cells. b) Processing of the cremaster muscle for cell culture
  • the cream muscle biopsy (2 cm 2 ) was cold transported in 50 ml of HBSS medium with 1% Fungizona and 1% Penicillin. The biopsy was stirred for muscle oxygenation. In a culture cabin, the biopsy was washed in cold HBSS and transferred with the tweezers to the p100 plate where it was cut into small fragments with sterilized tweezers and a surgical scalpel. The resulting fragments were transferred to a sterile tube with about 15 ml of filtered Type I collagenase and incubated at 37 ° C and stirred at 180 rpm for approximately 1-2h.
  • the collagenase disintegrated tissue was transferred to a sterile 50 ml tube to which 20 ml of proliferation medium was added to quench the collagenase.
  • the cell solution was subsequently filtered with a 40 pm Cell Strainer. To remove the collagenase, the solution was centrifuged at 1500 rpm 5 min at 23 ° C. The resulting pellet containing the multipotent precursor cells of the cremaster muscle was cultured and analyzed by different techniques. c) Cell culture for the formation of spheres
  • the pellet was resuspended in culture medium for non-adherent conditions, called proliferation medium (Neurobasal TM A medium supplemented with 2% supplement B27 50X, 1% L-glutamine 200 mM and 1% solution of penicillin / streptomycin 100X) and seeded in untreated plates.
  • proliferation medium Neurotrophic TM A medium supplemented with 2% supplement B27 50X, 1% L-glutamine 200 mM and 1% solution of penicillin / streptomycin 100X
  • growth factors LSGS, FGF2, EGF
  • the culture medium was changed every day for the first 3 days, and then do it on alternate days until 7 days. From day 1 after planting cell spheres could be seen. Washing was done by collecting the cells of each well in a sterile 50 ml tube and centrifuging at 1500 rpm for 5 min at RT. The pellet was resuspended again in 1 ml of proliferation medium and the cells were seeded in a new well with medium
  • the myospheres were disintegrated with 0.25% Trypsin-EDTA for 5 minutes at 37 ° C and then seeded the precursor cells on a natural extracellular matrix (ECM) that favors cell adhesion and differentiation (66, 88), composed of extract of basement membrane Cultrex® (2.77 mg / mL in final concentration), Netrin-4 (0.83 pg / mL), Netrin-G1a (0.83 pg / mL) and low molecular weight hyaluronic acid (2, 5 mg / mL) in PBS (pH 7.4).
  • ECM extracellular matrix
  • myotubes expressed specific striated muscle proteins such as MyHC and presented the characteristic striations of this muscular type.
  • PAX7 positive precursor cells possibly reserve satellite cells, were also observed.
  • RT-qPCR real-time quantitative PCR
  • Tedesco FS Dellavalle A, Diaz-Manera J, Messina G, Cossu G. Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. The Journal of clinical investigation. 2010; 120 (1): 11-9.
  • Emery AE The muscular dystrophies. Lancet 2002; 359 (9307): 687-95.
  • Tanyel FC Erdem S, Buyukpamukcu N
  • Tan E Cremaster muscle is not sexually dimorphic, but that from boys with undescended testis reflects alterations related to autonomic innervation. J Pediatr Surg. 2001; 36 (6): 877-80.

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Abstract

Les auteurs de la présente invention ont démontré que l'utilisation de cellules précurseurs pluripotentes (myogènes, vasculaires et neurales) isolées à partir du muscle crémaster présente des avantages pour la thérapie cellulaire chez des patients atteints d'incontinence urinaire ou anale avec lésion sphinctérienne ou dans le traitement de groupes musculaires de petite taille comme la distrophie musculaire oculopharyngée ou la dystrophie fascioscapulohumérale, en traitement unique ou combiné à d'autres principes actifs, ou associés à un dispositif médical.
PCT/ES2019/070167 2018-03-12 2019-03-12 Cellules précurseurs pluripotentes obtenues à partir du muscle crémaster et leur utilisation dans la mise au point de thérapies et médecine régénérative Ceased WO2019175459A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
NITTA M ET AL.: "Reconstitution of experimental neurogenic bladder dysfunction using skeletal muscle-derived multipotent stem cells", JOURNAL OF UROLOGY, vol. 181, no. 4, 1 April 2009 (2009-04-01), BALTIMORE, MD , US, pages 45 - 46, XP026008190, ISSN: 0022-5347, doi:10.1016/S0022-5347(09)60141-5 *
SHARIFIAGHDAS FARZANEH ET AL.: "Isolation of human adult stem cells from muscle biopsy for future treatment of urinary incontinence", UROLOGY JOURNAL IRAN WINTER 2011, vol. 8, no. 1, pages 54 - 59, XP55635208, ISSN: 1735-546X *
TAMAKI TETSURO ET AL.: "Therapeutic isolation and expansion of human skeletal muscle-derived stem cells for the use of muscle-nerve-blood vessel reconstitution", FRONTIERS IN PHYSIOLOGY JUN 2 2015, vol. 6, 6 February 2015 (2015-02-06), pages 1 - 16, XP55635197, ISSN: 1664-042X *
YAN WEI ET AL.: "Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture", EXPERIMENTAL CELL RESEARCH, vol. 317, no. 7, 15 January 2011 (2011-01-15), AMSTERDAM, NL . Ruas Jorge; Sven, pages 1016 - 1027, XP028178536, ISSN: 0014-4827, doi:10.1016/j.yexcr.2011.01.019 *

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