WO2019175459A1 - Multipotent precursor cells obtained from the cremaster muscle and use thereof to develop therapies and regenerative medicine - Google Patents

Multipotent precursor cells obtained from the cremaster muscle and use thereof to develop therapies and regenerative medicine 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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PCT/ES2019/070167
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Spanish (es)
French (fr)
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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
Administración General De La Comunidad Autónoma De Euskadi
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Publication of WO2019175459A1 publication Critical patent/WO2019175459A1/en

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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

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  • 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

The authors of the present invention propose that the use of multipotent precursor cells (myogenic, vascular and neural) isolated from the cremaster muscle has advantages for cell therapy in patients with urinary or anal incontinence with sphincter damage or for the treatment of small muscle groups, for example, in oculopharyngeal muscular dystrophy or facioscapulohumeral muscular dystrophy, the cells being used in a single treatment or a treatment combined with other active ingredients, or associated with a medical device.

Description

Células precursoras multipotentes obtenidas del músculo cremáster y su uso en el desarrollo de terapias y medicina regenerativa  Multipotent precursor cells obtained from the cremaster muscle and its use in the development of therapies and regenerative medicine
La presente invención se encuentra dentro del campo de la medicina regenerativa y de la terapia celular, y específicamente se refiere al método de aislamiento y expansión de células precursoras multipotentes obtenidas del músculo cremáster humano, y a sus usos terapéuticos. 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.
ESTADO DEL ARTE STATE OF ART
Las células madre adultas se caracterizan por sus propiedades de autorrenovación y diferenciación a distintos linajes (multipotencia) a lo largo de la vida del individuo. En general, son responsables del mantenimiento de los órganos y tejidos en homeostasis y de su reparación en respuesta al daño tisular, sea éste traumático o degenerativoAdult 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.
( (
En el músculo estriado adulto, las células madre miogénicas son conocidas como “células satélite”, debido a su posición superficial a las fibras musculares, aunque incluidas en la membrana basal que recubre las mismas (2). Sin embargo, el proceso de miogénesis es complejo y supone el paso de estas células por diversos estadios precursores (quiescencia, activación, proliferación y diferenciación) previos a su integración definitiva mediante su fusión con las fibras musculares en el marco del proceso regenerativo (3). Los marcadores reconocidos de las células satélite humanas incluyen Pax7, CD56, MyoD, M-cadherina y desmina, pero ninguno de ellos es considerado marcador específico de las mismas (4, 5). In adult striated muscle, 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). However, 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).
Por otro lado, el nicho de las células satélite es complejo: está compuesto por otras células precursoras, como los precursores fibro-adipogénicos o FAPs, que influyen en la respuesta a daño y en la regeneración compensatoria del tejido (6). Además, la célula satélite forma parte del espacio intersticial muscular y su nicho es adyacente a capilares y terminaciones nerviosas que, a su vez, influyen en la activación o quiescencia de las células satélite mediante señalización directa o paracrina, así como generando células precursoras de linaje vascular o neural en respuesta a daño muscular (7, 8). On the other hand, 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). In addition, 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).
Por todo ello, sería deseable la obtención y caracterización de los diversos tipos de células precursoras multipotentes existentes en el músculo esquelético y ser capaces de diseñar protocolos que permitan su utilización, por separado o en combinación, en estudios clínicos de terapia celular y de desarrollo de fármacos que modulen su activación. Se han publicado diversos protocolos de aislamiento de las células satélite humanas (o células precursoras miogénicas relacionadas) a partir de suspensiones celulares obtenidas del músculo esquelético, principalmente mediante la separación de subpoblaciones celulares en base a la expresión diferencial de marcadores de membrana. De hecho, esta ha sido un área especialmente activa de investigación en los últimos años. Así, Therefore, it would be desirable to obtain and characterize the various types of multipotent precursor cells existing in skeletal muscle and be able to design protocols that allow their use, separately or in combination, in clinical studies of cell therapy and development of drugs that modulate its activation. Various protocols for the isolation of human satellite cells (or related myogenic precursor cells) from cell suspensions obtained from skeletal muscle have been published, primarily by separating cellular subpopulations based on the differential expression of membrane markers. In fact, this has been an especially active area of research in recent years. So,
• Zheng y cois. definen una población “mioendotelial” CD45-• Zheng and cois. define a “myoendothelial” population CD45-
CD56+CD34+CD144+, que es Pax7+, supone el 0.4% de la suspensión celular inicial aislada de músculo esquelético humano, y que es superior en cuanto a su capacidad regenerativa en comparación a los mioblastos CD56+, cuando es trasplantada a músculo de ratón (9). 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).
• Distintos grupos de investigación han propuesto que las células mononucleadas CD133+ aisladas de músculo esquelético humano, aunque de naturaleza heterogénea, son capaces de generar células satélite tras su trasplante en ratón de manera más eficiente que los cultivos de mioblastos (10- 12). Un ensayo clínico Fase I demostró que las células CD133+ aisladas de músculo son seguras para su uso en seres humanos (13), pero el uso de esta población celular no se ha generalizado debido a que existen problemas en su expansión celular que deben ser resueltos de cara a un mayor desarrollo de su aplicación clínica (5).  • Different research groups have proposed that 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).
• Vauchez y cois proponen que las células musculares que no expresan el marcador CD34 y que presentan actividad aldehido deshidrogenasa (ADLH+), pueden ser una subpoblación celular progenitora de músculo, distinta de las células satélite (ya que son CD56-) y también distinta de las células CD133+ (ya que no expresan este marcador). La población ADLH+CD34- no cultivada es heterogénea, representa en torno al 3% de la suspensión celular inicial, y tras su aislamiento y trasplante directo a músculo de ratón (en ausencia de cultivo) demuestran propiedades regenerativas (14). Estos resultados han sido corroborados posteriormente por otros grupos de investigación (15, 16).  • Vauchez and cois propose that muscle cells that do not express the CD34 marker and that have aldehyde dehydrogenase (ADLH +) activity, may be a progenitor cell subpopulation of muscle, distinct from satellite cells (since they are CD56-) and also distinct from CD133 + cells (since they do not express this marker). The ADLH + CD34-uncultivated population is heterogeneous, represents around 3% of the initial cell suspension, and after isolation and direct transplantation to mouse muscle (in the absence of culture) demonstrate regenerative properties (14). These results have subsequently been corroborated by other research groups (15, 16).
• Pisani y cois describen dos subpoblaciones celulares miogénicas en músculo esquelético humano. Una, que identifican con las células satélite es definida como células CD34-CD15-CD56+. Otro precursor miogénico de carácter “intersticial” y no satélite (ya que es Pax7-) ni CD133+ (ya que no expresan este marcador) es definido como CD34+CD15-CD56+ (17). En otro estudio, proponen el uso de la fracción CD34- de células musculares para evitar la formación de grasa por las células tras su trasplante a músculo de ratones (18). El primer artículo carece de una validación del potencial regenerativo de estas poblaciones celulares en trasplantes in vivo, y el segundo estudio no enriquece suficientemente las poblaciones precursoras miogénicas. • Pisani and cois describe two myogenic cell subpopulations in human skeletal muscle. One, which they identify with satellite cells is defined as CD34-CD15-CD56 + cells. Another myogenic precursor of “interstitial” character and not satellite (since it is Pax7-) or CD133 + (since they do not express this marker) is defined as CD34 + CD15-CD56 + (17). In another study, they propose the use of the CD34- fraction of muscle cells to prevent the formation of fat by the cells after their transplantation into the muscle of mice (18). The first article lacks a validation of the regenerative potential of these cell populations in in vivo transplants, and the second study does not sufficiently enrich myogenic precursor populations.
• Lecourt y cois corroboran lo anterior, identificando una subpoblación CD56+ en cultivos de mioblastos que es miogénica y mutuamente excluyente con una subpoblación CD15+ que no es miogénica (19).  • Lecourt and cois corroborate the above, identifying a CD56 + subpopulation in myoblast cultures that is myogenic and mutually exclusive with a CD15 + subpopulation that is not myogenic (19).
• Castiglioni y cois proponen una estrategia de selección negativa para cinco marcadores (CD45, CD11b, GlyA, CD31 , CD34), seguida de selección positiva de las células CD56+ITGA7+ como estrategia de aislamiento de las células satélite de músculo esquelético fetal y adulto (20). En adultos, las células satélite así definidas suponen aproximadamente el 1.5% de la suspensión celular. Los autores calculan que existen unas 3600 células satélite por gramo de músculo, lo que a nivel práctico impide su aislamiento en cantidades suficientes para su uso en clínica. A nivel de marcadores, este estudio demuestra que las células satélite fetales expresan además CD144, CD146 y CD133, mientras que las células satélite adultas son CXCR4+.  • Castiglioni and cois propose a negative selection strategy for five markers (CD45, CD11b, GlyA, CD31, CD34), followed by positive selection of CD56 + ITGA7 + cells as a strategy for isolation of the fetal and adult skeletal muscle satellite cells ( twenty). In adults, the satellite cells so defined represent approximately 1.5% of the cell suspension. The authors estimate that there are about 3600 satellite cells per gram of muscle, which at a practical level prevents their isolation in sufficient quantities for clinical use. At the level of markers, this study demonstrates that fetal satellite cells also express CD144, CD146 and CD133, while adult satellite cells are CXCR4 +.
• En concordancia con lo anterior, Bareja y cois proponen la selección de células satélite humanas basada en la expresión de CD56 y CXCR4. Concretamente, seleccionan la población CD11b-CD31-CD34-CD45- CXCR4+CD56+ de músculo esquelético. Las células satélite así definidas suponen aproximadamente el 2% de la suspensión celular y son capaces de regenerar músculo in vivo tras su trasplante a músculo de ratón (21).  • In accordance with the above, Bareja and cois propose the selection of human satellite cells based on the expression of CD56 and CXCR4. Specifically, they select the population CD11b-CD31-CD34-CD45-CXCR4 + CD56 + of skeletal muscle. 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).
• Tamaki y cois proponen la selección de células precursoras miogénicas de músculos abdominales y de los miembros inferiores, basada en la combinación de marcadores CD34-CD45-CD29+ (22). Estas células fueron capaces de regenerar músculo en el trasplante in vivo a músculo de ratón. Sin embargo, la proporción de células positivas para CD29 es demasiado alta para ser una población homogénea, en torno a un 10-12% del total, lo que indica que el protocolo de purificación de esta subpoblación podría refinarse.  • Tamaki and cois propose the selection of myogenic precursor cells of abdominal muscles and lower limbs, based on the combination of markers CD34-CD45-CD29 + (22). These cells were able to regenerate muscle in transplantation in vivo to mouse muscle. However, the proportion of CD29 positive cells is too high to be a homogeneous population, around 10-12% of the total, indicating that the purification protocol of this subpopulation could be refined.
• En esta línea, Xu y cois proponen que, tras una selección negativa para eliminar las células endoteliales (CD31+) y células del sistema inmune (CD45+), la combinación de marcadores CD56+/CD29+ selecciona específicamente las células satélite de diversos músculos esqueléticos humanos, que suponen aproximadamente el 1 % de la suspensión celular inicial (23). En ensayos de trasplante a ratones de las células recién aisladas (no cultivadas), esta subpoblación es la única capaz de generar fibras regenerativas, dando lugar a células satélite funcionales que colonizan el nicho en la fibra muscular receptora y son capaces de responder a un segundo daño en el tejido. Siendo esta aproximación altamente prometedora, hay que tener en cuenta que el número de células obtenido en la realidad fue 40-100 veces menor al que se esperaba en base al cálculo teórico de las células satélite preexistentes. • Along these lines, Xu and cois propose that, after a negative selection to eliminate endothelial cells (CD31 +) and immune system cells (CD45 +), the combination of CD56 + / CD29 + markers specifically selects the satellite cells of various human skeletal muscles, which represent approximately 1% of the initial cell suspension (23). In mice transplantation assays of newly isolated (uncultivated) cells, this subpopulation is the only one capable of generating regenerative fibers, giving rise to functional satellite cells that colonize the niche in the receiving muscle fiber and are able to respond to a second tissue damage. Being this highly promising approach, it should be borne in mind that the number of cells actually obtained was 40-100 times less than expected based on the theoretical calculation of pre-existing satellite cells.
• En concordancia parcial con lo anterior, Charville y cois proponen que las células satélite humanas se pueden aislar de diversos músculos con la combinación de marcadores CD31-CD34-CD45-CD29+EGFR+ (24). Este estudio propone la inhibición de la ruta de p38MAPK para lograr una expansión de las células satélite indiferenciadas en el cultivo in vitro, que además aumenta posteriormente entre 3 y 7 veces la capacidad de regeneración muscular al ser trasplantadas al músculo de ratón. Los autores atribuyen estas mejoras a la disminución de la diferenciación y el rejuvenecimiento de las células satélite al ser cultivadas en presencia de un inhibidor de p38MAPK. • In partial agreement with the above, Charville and cois propose that human satellite cells can be isolated from various muscles with the combination of markers CD31-CD34-CD45-CD29 + EGFR + (24). This study proposes the inhibition of the p38MAPK pathway to achieve an expansion of undifferentiated satellite cells in in vitro culture, which also subsequently increases the regeneration capacity between 3 and 7 times when they are transplanted to the mouse muscle. The authors attribute these improvements to the decrease in differentiation and rejuvenation of satellite cells when cultured in the presence of a p38MAPK inhibitor.
• Alexander y cois proponen que CD82 es un marcador expresado por las células satélite humanas y que puede ser utilizado para su aislamiento, aunque no es específico de las mismas porque sigue expresándose en células precursoras miogénicas activadas o en proceso de diferenciación. Además, su expresión está disminuida en pacientes de distrofia muscular, lo que comprometería su posible uso en terapia en algunos tipos de enfermedades (25). • Alexander and cois propose that 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).
• En concordancia parcial con lo anterior, Uezumi y cois proponen que, partiendo de cultivos de células CD56+ humanas, las células positivas para los marcadores de membrana CD82, CD274 y CD318 son las células satélite sublaminares, mientras que CD201 es marcador del precursor adipogénico residente en músculo ó FAP (26). Las células satélite aisladas a partir de cultivos de mioblastos CD56+ son definidas en este estudio como CD56+CD82+CD318+PDGFRa-CD201-  • In partial agreement with the above, Uezumi and cois propose that, based on cultures of human CD56 + cells, the positive cells for the membrane markers CD82, CD274 and CD318 are the sublaminar satellite cells, while CD201 is a marker of the resident adipogenic precursor in muscle or FAP (26). Satellite cells isolated from CD56 + myoblast cultures are defined in this study as CD56 + CD82 + CD318 + PDGFRa-CD201-
A pesar de todos estos avances recientes, las células satélite aún presentan importantes problemas de cara a su posible aplicación clínica como medicamento de terapia celular. El número de células satélite que se obtiene de las biopsias musculares de pequeño tamaño (típicamente de 50-100 mg) que se utilizan normalmente en clínica es muy limitado. Se calcula que hay entre 500 y 1000 células satélite por milímetro cúbico de fibra en el músculo humano, aunque su número es variable entre los distintos grupos musculares en función del área y densidad de las miofibras (23). Además, estas células se expanden insuficientemente ex vivo o pierden sus características de célula madre como consecuencia del cultivo in vitro. Esto se ha demostrado tanto en células satélite de ratón (27, 28) como humanas (29, 30). Es más, los protocolos de aislamiento y caracterización de las células satélite humanas se encuentran todavía en desarrollo, a diferencia del ratón donde se conoce mejor su biología (31). Despite all these recent advances, 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). In addition, 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. Moreover, the isolation and characterization protocols of human satellite cells are still in development, unlike the mouse where its biology is best known (31).
Por estas razones, los cultivos celulares denominados como“mioblastos” han sido los más usados en clínica hasta el momento. Los mioblastos (32) son una mezcla heterogénea de células en suspensión obtenidas de biopsias musculares, normalmente utilizadas directamente (sin purificar), o semi-purificadas mediante un mínimo enriquecimiento basado en la selección positiva de las células que expresan el antígeno de membrana CD56 (33). En general, los investigadores del campo consideran que los mioblastos son células precursoras miogénicas, en un estadio intermedio entre las células satélite y los miocitos terminalmente diferenciados, aunque esto es difícil de precisar dada la heterogeneidad de estos cultivos (incluidos los obtenidos a partir de la fracción celular CD56+). Los cultivos de mioblastos así obtenidos se expanden in vitro con relativa facilidad (34, 35), por lo que han sido utilizados en diversos ensayos clínicos en indicaciones relacionadas con músculo esquelético y cardiaco, en los que los resultados obtenidos han sido decepcionantes (36-40). For these reasons, cell cultures called "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). In general, field researchers consider that 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).
Los motivos del fracaso clínico de los mioblastos pueden ser múltiples, pero están probablemente relacionados con las condiciones de expansión celular (41 , 42). Cuando los medios y condiciones de cultivo favorecen la proliferación, es fácil que provoquen también la excesiva diferenciación celular, lo que resulta en baja supervivencia, capacidad migratoria y fusogenicidad cuando estas células expandidas in vitro son trasplantadas a zonas de daño muscular in vivo (5, 43). Ejemplos de señalizaciones implicadas en la regulación de la expansión vs diferenciación de las células satélite de ratón incluyen los niveles de oxígeno presentes en la cámara de cultivo (44), cambios en el metabolismo celular (45-47), las efrinas y sus receptores (48), la ruta de los FGF (49), la ruta de Notch (50), SOX7 (51), TAZ/YA P (52) y la ruta TGFbeta/Smad (53), entre otros. Aun así, es importante señalar que la gran mayoría de estudios al respecto están realizados en modelos animales y su traslación a las células humanas es incierta (54). The reasons for the clinical failure of 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).
En el ratón, es conocido que los cultivos de mioblastos retienen una subpoblación celular minoritaria (de ciclo celular más lento), que además es la responsable de la toma del trasplante una vez inyectados los cultivos en el animal receptor, mientras que el resto de células entra en apoptosis en las primeras horas tras el trasplante (55). La pre-selección de estas células de ciclo lento previo al trasplante mejoró mucho los resultados del mismo (56). In the mouse, it is known that 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).
Con respecto a los cultivos de mioblastos humanos, se sabe que estos cultivos heterogéneos son capaces de regenerar el músculo (57) e incluso generar células satélite humanas tras su trasplante a ratones (58). Laumonier y cois proponen que los cultivos de mioblastos humanos retienen una población de células satélite“reserva”, identificada como CD45-CD34-CD144-CD56+CD146+ tras 5-7 días de cultivo (59). Estas células son Pax7+MyoD-Myf5+ (es decir, similares a las células satélite quiescentes) y muestran mejor supervivencia que los mioblastos tras su trasplante a ratón. 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.
En el mejor de los casos, se cree que los cultivos de mioblastos pueden tener éxito en la reparación de músculos pequeños como los afectados en la distrofia muscular oculofaríngea. Sin embargo, en los ensayos clínicos en marcha se están inyectando cantidades muy elevadas de células (cientos de millones; (60)), lo que indica que el potencial de regeneración de estos cultivos es claramente mejorable y que sería deseable obtener poblaciones celulares más puras que permitan reducir la expansión de los productos de terapia celular (para evitar los riesgos asociados a los largos periodos de expansión requeridos). Alternativamente o en paralelo, podría ser útil también la co-inyección de otras células precursoras que faciliten la regeneración, revascularización y reinnervación del área dañada. In the best case, it is believed that myoblast cultures may be successful in repairing small muscles such as those affected in the oculopharyngeal muscular dystrophy. However, 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). Alternatively or in parallel, co-injection of other precursor cells that facilitate regeneration, revascularization and reinnervation of the damaged area could also be useful.
Con estos antecedentes, es fácil comprender que determinar las condiciones ideales de aislamiento y expansión de precursores miogénicos humanos (bien las propias células satélite musculares o bien precursores intermedios entre éstas y los mioblastos), así como de otros precursores multipotentes presentes en el músculo humano, es un objetivo de gran importancia para la obtención de una(s) población(es) celular(es) que tenga(n) utilidad clínica en regeneración miogénica. With this background, it is easy to understand that determining the ideal conditions of isolation and expansion of human myogenic precursors (either the muscle satellite cells themselves or intermediate precursors between them and myoblasts), as well as other multipotent precursors present in the human muscle, It is an objective of great importance for obtaining a cell population (s) that has clinical utility in myogenic regeneration.
De manera consistente con los estudios previamente mencionados sobre la actividad aldehido deshidrogenasa en células satélite, la adición de ácido retinoico al medio de cultivo de mioblastos humanos parece favorecer la retención de características de célula madre y la ausencia de diferenciación gracias a un menor estrés oxidativo (61). En mioblastos humanos, se ha demostrado además que la inhibición de la ruta de TGFbeta por LY364947 y el uso de Geraldol y Bromopride aumentan la proliferación celular, mientras que los compuestos Pratol, Limonin e Idirubin aumentan la diferenciación (62). Existen también aproximaciones distintas al cultivo clásico de mioblastos, en condiciones de adhesión a un soporte bidimensional. Así, el crecimiento de células derivadas de músculo de ratón y rata en cultivo tridimensional en forma de mioesferas flotantes enriquece las mismas en células Pax7+MyoD+, potencialmente células satélite musculares, que se diferenciaron in vitro a células contráctiles mononucleadas y contribuyeron a la regeneración de fibras musculares tras su trasplante a músculo de ratón (63). Estudios posteriores han demostrado que las mioesferas de ratón contienen al menos dos subpoblaciones celulares, una célula precursora miogénica (posiblemente célula satélite) que es ITGA7+Myf5+MyoD+Pax7+ y otra célula precursora no miogénica (posiblemente el precursor fibroadipogénico- FAP) caracterizada como PDGFRa+Sca-1+ (64, 65). Es importante señalar que ambas células precursoras son clave para el correcto funcionamiento del músculo, tanto en homeostasis como en regeneración, por lo que el cultivo de mioesferas tendría la ventaja añadida de proveer de al menos dos tipos de células precursoras, en vez de uno. Consistent with the previously mentioned studies on aldehyde dehydrogenase activity in satellite cells, the addition of retinoic acid to the culture medium of human myoblasts seems to favor the retention of stem cell characteristics and the absence of differentiation due to a lower oxidative stress ( 61). In human myoblasts, it has also been shown that inhibition of the TGFbeta pathway by LY364947 and the use of Geraldol and Bromopride increase cell proliferation, while the compounds Pratol, Limonin and Idirubin increase differentiation (62). There are also different approaches to the classical culture of myoblasts, under conditions of adhesion to a two-dimensional support. Thus, the growth of 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). Subsequent studies have shown that 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.
De hecho, es probable que el cultivo de mioesferas contenga también un tercer tipo de células precursoras, los precursores neurales, tal y como hemos demostrado previamente en la caracterización de esferas miogénicas aisladas de la dermis de ratón (66). In fact, it is likely that 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).
Aun así, el estudio de las miosferas derivadas de músculo humano está en su infancia. El primer estudio publicado con miosferas humanas presenta una metodología pobremente descrita, pero propone la existencia en las miosferas de una población de células precursoras“mesenquimales” definida como CD31-CD34-CD45-CD56-CD117- CD29+CD73+CD90+CD105+, posiblemente no miogénica en trasplante a músculo de ratón, aunque este hecho no llegó a investigarse (67). Un segundo artículo define la existencia de una célula precursora miogénica en mioesferas derivadas de biopsias obtenidas del músculo omohioideo en pacientes con carcinoma de cabeza y cuello (68). Las biopsias fueron de un mínimo de 100 mg para poder establecer el cultivo esferogénico, y se pudieron expandir durante 20 semanas (18 pases), aunque los cultivos comenzaron a crecer más lento tras la semana 16. Estas células precursoras, posiblemente células satélite se definen como CD34-CD45-Pax7+CD56+ALDH1+ y, sorprendentemente, se definen también como Oct3/4+Nanog+Sox2+, indicando su estado de pluripotencia en estas condiciones de cultivo. Sólo el 50% de estas células expresaron desmina y MyoD en pases tempranos, demostrando la heterogeneidad de la subpoblación a estudio. Una causa adicional de preocupación con este estudio es la posible contaminación con células cancerosas. No existen más publicaciones, hasta donde conocemos, describiendo aproximaciones basadas en el cultivo tridimensional de miosferas a partir de músculo esquelético humano. Even so, the study of myospheres derived from human muscle is in its infancy. The first study published with human myospheres presents a poorly described methodology, but proposes the existence in the miospheres of a population of "mesenchymal" precursor cells defined as CD31-CD34-CD45-CD56-CD117-CD29 + CD73 + CD90 + CD105 +, possibly Non-myogenic in mouse muscle transplantation, although this fact was not investigated (67). A second article defines the existence of a myogenic precursor cell in myospheres derived from biopsies obtained from the omohyoid muscle in patients with head and neck carcinoma (68). 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. There are no more publications, as far as we know, describing approaches based on the three-dimensional culture of myospheres from human skeletal muscle.
La regeneración muscular mediante protocolos de terapia celular basados en células precursoras miogénicas, vasculogénicas y que promuevan la reinnervación puede tener aplicación en diversas disfunciones del músculo esquelético y cardiaco, como las miopatías congénitas (69), las distrofias musculares y otras enfermedades degenerativas neuromusculares (70), la disfunción cardiaca (71), la pérdida de masa muscular volumétrica, la caquexia o la sarcopenia (72). 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).
Sin embargo, algunas de estas enfermedades afectan a grandes masas de tejido muscular y esto compromete la viabilidad de su aproximación terapéutica por terapia celular. Algunos investigadores consideran que el tratamiento de grupos musculares de pequeño tamaño, como los afectados en la distrofia muscular oculofaríngea o la distrofia facioescapulohumeral, puede tener mayores posibilidades de éxito (5, 43). En este sentido, las deficiencias del esfínter urinario (73) y anal (74) pueden ser una excelente diana para este tipo de terapias, ya que el volumen de músculo a regenerar en los esfínteres es relativamente pequeño (5, 75) y el déficit muscular es accesible mediante aproximaciones quirúrgicas mínimamente invasivas. Para ello es necesario establecer los modelos adecuados de enfermedad, dada la gran variabilidad asociada a la patología observada en clínica (76). However, some of these diseases affect large masses of muscle tissue and this compromises the viability of their therapeutic approach by cell therapy. Some researchers consider that the treatment of small muscle groups, such as those affected in the oculopharyngeal muscular dystrophy or the facioscapulohumeral dystrophy, may have a greater chance of success (5, 43). In this sense, deficiencies of the urinary sphincter (73) and anal (74) can be an excellent target for this type of therapy, since the volume of muscle to regenerate in the sphincters is relatively small (5, 75) and the deficit Muscle is accessible by minimally invasive surgical approaches. For this, it is necessary to establish the appropriate disease models, given the great variability associated with the pathology observed in the clinic (76).
Es importante tener en cuenta que las células satélite podrían presentar identidades divergentes en base a su localización axial/anatómica o el tipo de fibra muscular del que provienen (3). Así, por ejemplo, el represor transcripcional Proxl es característico de las fibras musculares lentas y se expresa en las células satélite (77). En los protocolos clínicos publicados hasta el momento, las células miogénicas se han aislado de músculos diversos y no necesariamente idénticos o próximos (desde un punto de vista anatómico o de biología del desarrollo) a los del área prevista de aplicación (5). Sin embargo, no es descartable que el uso de células precursoras obtenidas de regiones similares a las regiones diana pueda tener ventajas asociadas en lo que respecta a la funcionalidad, capacidad de adaptación o supervivencia de las mismas. Algunos ejemplos similares en este sentido pueden ser (a) la obtención de mioblastos CD56+ del músculo piramidal en pacientes que van a sufrir una prostatectomía radical (78) y (b) la extracción de mioblastos del músculo recto abdominal (79), de manera que puedan ser utilizados posteriormente en casos de incontinencia urinaria. La incontinencia urinaria (IU) es una patología prevalente, que afecta a más de 200 millones de mujeres y a un 17% de varones en todo el mundo; aunque su diagnóstico puede estar infraestimado. Datos en modelos animales sugieren que la inyección de células precursoras puede mejorar la función debilitada del esfínter (75). De hecho, los ensayos clínicos que han utilizado inyección autóloga de células precursoras musculares obtenidas a partir de biopsias de músculo demuestran que esta terapia es segura y presenta resultados aceptables. Sin embargo, hasta ahora la terapia celular para la regeneración del esfínter urinario no ha logrado consolidarse en la práctica clínica, ya que la estandarización de esta metodología experimental podría ser compleja. Por lo tanto, son necesarios nuevos enfoques de investigación y perspectivas para garantizar el uso de esta terapia como una opción de tratamiento real (73, 80). Es importante señalar que las células precursoras musculares utilizadas hasta el momento han sido cultivos de mioblastos autólogos expandidos. Por lo tanto, sería de esperar la existencia de porcentajes relativamente bajos de células satélite (o incluso células precursoras en estadios intermedios de diferenciación) en las suspensiones celulares heterogéneas utilizadas para tratar a estos pacientes. Por otro lado, los cultivos de mioblastos carecen de precursores vasculares y neurales que promuevan la revascularización y reinnervación del nuevo tejido. It is important to keep in mind that satellite cells could present divergent identities based on their axial / anatomical location or the type of muscle fiber they come from (3). Thus, for example, the Proxl transcriptional repressor is characteristic of slow muscle fibers and is expressed in satellite cells (77). In the clinical protocols published so far, 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). However, it is not ruled out that 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. However, until now, cell therapy for urinary sphincter regeneration has not been consolidated in clinical practice, since the standardization of this experimental methodology could be complex. Therefore, new research approaches and perspectives are necessary to ensure the use of this therapy as a real treatment option (73, 80). It is important to note that the muscle precursor cells used so far have been expanded autologous myoblast cultures. Therefore, the existence of relatively low percentages of satellite cells (or even precursor cells in intermediate stages of differentiation) in the heterogeneous cell suspensions used to treat these patients would be expected. On the other hand, myoblast cultures lack vascular and neural precursors that promote revascularization and reinnervation of the new tissue.
La terapia celular aparece como una oportunidad única para estas enfermedades degenerativas, por lo que obtener una célula humana o mezcla de células con capacidad miogénica y regenerativa, y que se pueda trasplantar de manera autóloga o alogénica (en presencia de un régimen inmunosupresor), supondría un avance para el tratamiento específico de dichas enfermedades. Cell therapy appears as a unique opportunity for these degenerative diseases, so obtaining a human cell or mixture of cells with myogenic and regenerative capacity, and that can be transplanted autologously or allogeneically (in the presence of an immunosuppressive regimen), would be an advance for the specific treatment of these diseases.
BREVE DESCRIPCIÓN DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
Los autores de la presente invención proponen que el empleo de células precursoras multipotentes (miogénicas, vasculares y neurales) aisladas a partir del músculo cremáster presenta ventajas para la terapia celular en pacientes de incontinencia urinaria o anal con daño esfinteriano o en el tratamiento de grupos musculares de pequeño tamaño tales como la distrofia muscular oculofaríngea o la distrofia facioescapulohumeral, en tratamiento único o combinado con otros principios activos, o asociados a un dispositivo médico. 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.
El cremáster es un músculo estriado (aunque carece de inserción en tendón) y de control voluntario que está entremezclado con abundantes fibras de tipo muscular liso. Contiene predominantemente fibras de tipo 1 (lento), aunque también algunas de tipo II B (muy rápidas), y cuya función en el adulto es contribuir a la termorregulación y a la protección de los testículos como una de las túnicas que forman el escroto. Gracias al reflejo cremastérico, se conocen bien sus propiedades electrofisiológicas. El músculo está densamente innervado y presenta numerosas placas motoras, lo que explica sus abundantes descargas espontáneas. En un estudio realizado en niños y niñas con hernia inguinal, no se observaron dimorfismos sexuales en el músculo cremáster entre ambos sexos, excepto el mayor diámetro de las fibras musculares en el género masculino, que es común a otros grupos musculares. En el desarrollo embrionario, el cremáster se genera a partir del gubernáculo, independientemente del músculo interno oblicuo del abdomen, siendo clave en el descenso testicular. Algunos autores proponen la transdiferenciación del músculo estriado cremastérico a partir de fibras de músculo liso, lo que puede ser común a otros músculos del tracto genitourinario como el rabdoesfínter. 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. In a study conducted in boys and girls with inguinal hernia, no sexual dimorphisms were observed in the cremaster muscle between both sexes, except for the larger diameter of the muscle fibers in the male gender, which is common to other muscle groups. In embryonic development, the cremaster is generated from the tubercle, regardless of the oblique internal muscle of the abdomen, being key in the testicular descent. Some authors propose transdifferentiation of the crematorial striated muscle from smooth muscle fibers, which may be common to other muscles of the genitourinary tract such as the rhabdoesphincter.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1. Caracterización del músculo cremáster masculino. A. Localización anatómica del músculo cremáster. Se encuadra la zona de toma de biopsias. B. Sección histológica con tinción de hematoxilina-eosina donde se observan los paquetes de fibras musculares estriadas y lisas (b’, detalle a mayor aumento). C-D. Por inmunofluorescencia, se observa que las fibras estriadas miosina positivas (MyHC+, verde) están rodeadas por membrana basal (Laminin+, rojo). Los núcleos se muestran en azul. E-H. Análisis de la composición de miosina en las fibras de tipo I (lentas, verde) y II (rápidas, verde). La laminina se muestra en rojo y los núcleos en azul. I-J. Cuantificación del número de fibras regenerativas mediante mionúcleos centrales. K-L. Cuantificación del número de fibras regenerativas mediante la detección de la expresión de MyHC3 embrionaria (verde). M-N. Cuantificación del número de células satélite in situ (Pax7+; verde). Figure 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. Quantification of the number of regenerative fibers by central myionuclei. K-L Quantification of the number of regenerative fibers by detecting the expression of embryonic MyHC3 (green). M-N. Quantification of the number of satellite cells in situ (Pax7 +; green).
Figura 2. Células precursoras multipotentes presentes en dermoesferas de ratón. A. Aspecto del cultivo en suspensión en esferas derivadas de la dermis del ratón. B-E. Tras su disgregación y adhesión al sustrato, se observa que las esferas contienen células precursoras vasculares de aspecto fusiforme (B), células adiposas (C), células precursoras neurales, con largas prolongaciones (D) y células precursoras miogénicas, que en algunos casos comienzan a formar sincitios multinucleados (E). Figura 3. Detección de células precursoras miogénicas del músculo cremáster en mioesferas. A. Esquema temporal del cultivo celular en condiciones no adherentes. B-C. Imagen de contraste de fases de un cultivo celular a partir de biopsia de cremáster a día 0 y día 7. La flecha en el panel B señala restos no disociados de músculo. D-F. Análisis de la expresión de marcadores miogénicos por inmunofluorescencia. Se detectó tinción (en verde) para los marcadores Pax7 (D), MyoD1 (E) y MyHC (F). Los núcleos se muestran en azul. Figure 2. Multipotent precursor cells present in mouse dermospheres. A. Aspect of the suspension culture in spheres derived from the dermis of the mouse. BE. After disintegration and adhesion to the substrate, it is observed that the spheres contain spindle-shaped vascular precursor cells (B), fat cells (C), neural precursor cells, with long extensions (D) and myogenic precursor cells, which in some cases begin to form multinucleated syncytia (E). Figure 3. Detection of myogenic precursor cells of the cremester muscle in myospheres. A. Temporary scheme of cell culture under non-adherent conditions. BC Contrast image of phases of a cell culture from a cremaster biopsy at day 0 and day 7. 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.
Figura 4. Detección de células precursoras neurogénicas y vasculogénicas del músculo cremáster en mioesferas. A-E. Análisis de la expresión de marcadores de precursores neurogénicos y vasculogénicos por inmunofluorescencia. Se detectó tinción en verde para el marcador CD56 (A), en rojo para p75NTR (B) y en azul (C) para los núcleos (DAPI). El panel D muestra los paneles A-B superpuestos, y E la superposición de A-B-C. Los siguientes símbolos muestran las células neurogénicas (p75NTR+CD56+, asterisco), las células vasculogénicas (p75NTR+CD56-, flecha) y las células p75NTR-CD56+ (cabeza de flecha). 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).
Figura 5. Diferenciación in vitro de las células precursoras del cremáster en condiciones miogénicas. A. Diagrama temporal de formación de miotubos a partir de esferas disgregadas. B. Imagen en contraste de fases del cultivo de esferas disgregadas a día 0 en medio de diferenciación. C. Imagen en contraste de fase de los miotubos multinucleados formados tras 7 días en cultivo. D-G. Caracterización de los miotubos mediante inmunofluorescencia con los anticuerpos anti-MyHC (verde) y Pax7 (verde). Los núcleos se muestran en azul. H. Análisis de la expresión de genes determinantes para la miogénesis ( Pax7 , Myf5, MyoD1, Myogenin, MyHC3 y MyHC2 ) mediante retrotranscripción seguida de PCR cuantitativa a tiempo real (RT-qPCR). Se muestran los resultados obtenidos de manera relativa al gen control Tbp. Figure 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.
Figura 6. Capacidad regenerativa in vivo de las células precursoras del cremáster. A. Esquema experimental. B. Inmunofluorescencia anti-hLaminA/C (específica de músculo humano, verde). Los núcleos se muestran en azul. C. Inmunofluorescencia anti-hLaminA/C (verde) y anti-hDystrophin (también específica de músculo humano, rojo). E-F. Cuantificación del número de células hLaminA/C+ (verdes) que co-expresa el marcador de las células satélite Pax7 (rojo). Los núcleos se muestran en azul. DESCRIPCIÓN DE LA INVENCIÓN Figure 6. Regenerative capacity in vivo of the precursor cells of the cremaster. A. Experimental scheme. B. Anti-hLaminA / C immunofluorescence (specific for human muscle, green). The cores are shown in blue. C. Immunofluorescence anti-hLaminA / C (green) and anti-hDystrophin (also specific to human muscle, red). EF. Quantification of the number of hLaminA / C + cells (green) that co-expresses the Pax7 satellite cell marker (red). The cores are shown in blue. DESCRIPTION OF THE INVENTION
Los autores de la presente invención proponen que el empleo de células precursoras multipotentes (miogénicas, vasculares y neurales) aisladas a partir del músculo cremáster presenta ventajas para la terapia celular en pacientes de incontinencia urinaria o anal con daño esfinteriano o en el tratamiento de grupos musculares de pequeño tamaño tales como la distrofia muscular oculofaríngea o la distrofia facioescapulohumeral, en tratamiento único o combinado con otros principios activos, o asociados a un dispositivo médico. 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.
Hasta el momento no se ha empleado el músculo cremáster como fuente de células precursoras. La principal función del cremáster es la bajada del testículo hasta el anillo inguinal mediante su contracción, por lo que el obtener una biopsia no supondría a los pacientes adultos un importante daño funcional en dicha zona y podría emplearse como fuente de células multipotentes para el estudio y tratamiento de pacientes con desórdenes traumáticos o degenerativos que afecten a grupos musculares. So far, 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.
Por lo tanto, los autores de la presente invención muestran que el músculo cremáster representa una fuente valiosa para aislar células precursoras multipotentes con capacidad de sustituir estructuralmente y bioquímicamente: i) la pared muscular del esfínter urinario, anal, u otros órganos o tejidos que incluyan fibras o haces de músculo estriado, ii) sustitución de células miogénicas del esfínter urinario u anal dañadas o afuncionales o del músculo estriado dañado en esta u otras patologías musculares, iii) la recuperación del músculo estriado, del esfínter urinario, o de otros órganos, como por ejemplo el esfínter anal, o puede tener aplicación además en diversas disfunciones del músculo esquelético y cardiaco, como las miopatías congénitas, las distrofias musculares y otras enfermedades degenerativas neuromusculares, la disfunción cardiaca, la pérdida de masa muscular volumétrica, la caquexia o la sarcopenia, iv) sustitución de células miogénicas utilizadas como parte constituyente de órganos generados por técnicas de ingeniería tisular como la uretra, tráquea y otros tejidos y órganos, v) sustitución de células vasculogénicas que promuevan la revascularización del tejido dañado, vi) sustitución de células neurogénicas que promuevan la reinnervación del tejido dañado; Therefore, the authors of the present invention show that 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, v) replacement of vasculogenic cells that promote the revascularization of damaged tissue; vi) replacement of neurogenic cells that promote the reinnervation of damaged tissue;
Además, desarrollan una metodología para lograr la colonización y diferenciación miogénica completa en formulaciones basadas en diferentes biomateriales tales como agarosa, alginatos, andamios basados en matriz extracelular, colágeno, Cultrex, ferrogeles, fibrina, fibrina/agarosa, fibrinógeno, fibronectina, gelatina, ácido hialurónico (HA), hidrogeles, laminina, Matrigel, plasma, plasma rico en factores de crecimiento, policaprolactona (PCL), ácido poliglicólico (PGA), polietilénglicol (PEG), PEG- fibrinógeno, poliglicerol sebacato (PGS), ácido poliláctico-glicólico (PLGA), ácido poliláctico (PLLA), quitosano (CHT), polímeros acrílicos como PEA, PHEA, PMA y otros, láminas de seda, matriz extracelular de órganos descelularizados como el músculo y otros tejidos y órganos como la submucosa, utilizando células precursoras aisladas a partir del músculo cremáster. Esta lista se proporciona sólo a modo de ilustración, y no se pretende que sea limitante. In addition, they develop a methodology to achieve complete myogenic colonization and differentiation in formulations based on different biomaterials such as agarose, alginates, scaffolds based on extracellular matrix, collagen, Cultrex, ferrogels, fibrin, fibrin / agarose, fibrinogen, fibronectin, gelatin, acid hyaluronic (HA), hydrogels, laminin, Matrigel, plasma, growth-rich plasma, polycaprolactone (PCL), polyglycolic acid (PGA), polyethylene glycol (PEG), PEG-fibrinogen, polyglycerol sebacate (PGS), polylactic-glycolic acid (PLGA), polylactic acid (PLLA), chitosan (CHT), acrylic polymers such as PEA, PHEA, PMA and others, silk sheets, extracellular matrix of decellularized organs such as muscle and other tissues and organs such as submucosa, using precursor cells isolated from the cremaster muscle. This list is provided by way of illustration only, and is not intended to be limiting.
Los autores de la presente invención son los primeros que han desarrollado un método para obtener células precursoras multipotentes aisladas del músculo cremáster. The authors of the present invention are the first who have developed a method to obtain multipotent precursor cells isolated from the cremaster muscle.
CÉLULAS Y POBLACIONES CELULARES DE LA INVENCIÓN CELLS AND CELLULAR POPULATIONS OF THE INVENTION
Por tanto, un primer aspecto de la invención se refiere a una composición que comprende una población celular de células precursoras multipotentes derivadas o provenientes del músculo cremáster humano, de ahora en adelante composición de células precursoras multipotentes de la invención, donde dicha población, a su vez, representa una población heterogénea de células que incluye diversos tipos de células precursoras, tal y como se ilustra en la figura 2, como células precursoras miogénicas, células precursoras vasculogénicas, y células precursoras neurogénicas. Se hace notar que la población celular referida, de ahora en adelante población celular de células precursoras multipotentes, debe comprender al menos una célula precursora multipotente derivada o proveniente del músculo cremáster humano. En una realización preferida dicha población celular comprende al menos un 20%, preferiblemente un 30%, más preferiblemente un 40%, y aún más preferiblemente un 50%, 60%, 80%, 90%, 95%, o un 99% de células precursoras multipotentes derivadas o provenientes del músculo cremáster humano. Therefore, a first aspect of the invention relates to 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. It is noted that 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. In a preferred embodiment 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.
Para conseguir dichas células del primer aspecto de la invención, se parte de biopsias de músculo cremáster, se trocea dicha biopsia de tejido en fragmentos pequeños y, una vez disgregado el tejido, se obtienen las células precursoras multipotentes del músculo cremáster, y a continuación se cultivan (ver ejemplos de la invención). In order to achieve said cells of the first aspect of the invention, 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).
Un segundo aspecto de la invención se refiere a una composición que comprende mioesferas obtenidas de cultivos de una población celular de células aisladas del músculo cremáster del primer aspecto de la invención. Para ello y tal y como se ilustra en los ejemplos de la invención, se re-suspende la composición obtenida del primer aspecto de la invención en medio de cultivo para condiciones no adherentes, llamado medio de proliferación (medio Neurobasal™ A suplementado con 2% de suplemento B27 50X, 1% de L-glutamina 200 mM y 1 % de solución de penicilina/estreptomicina 100X) y se siembran en placas no tratadas para obtener un cultivo de mioesferas. El precipitado celular resultante del aislamiento celular se resuspende en 1 ml_ de medio para cultivo en suspensión y se realiza un recuento y control de la viabilidad celular antes de traspasarlo a un pocilio de las placas no tratadas de 6 pocilios, donde se añade más medio para cultivo en suspensión hasta 5 ml_. Si el recuento de células viables resulta ser menor que 500.000 células/mL, estas se traspasan a un pocilio de menor tamaño, de placas no tratadas de 12 pocilios, y se ajustan los volúmenes de medio y de factores en proporción a lo establecido. Estas placas evitan la adhesión de las células y favorecen la formación de esferas celulares. En este punto se añaden directamente al pocilio en las concentraciones indicadas los factores de crecimiento (Suplemento de crecimiento bajo en suero (LSGS; concentración final del 2% (1X), Factor de crecimiento fibroblástico básico recombinante de rata (rrFGF2; 80 ng/ml_), Factor de crecimiento epidérmico recombinante de rata (rrEGF; 40 ng/ml_). Los primeros 3 días se cambia el medio por completo mediante centrifugación (5 minutos a 1500 rpm) para eliminar restos celulares y seleccionar las células viables, y se añaden los factores de crecimiento de nuevo cada vez. Después, estos lavados se hacen cada 2 días y el cultivo se mantiene hasta los 7 o 9 días, cuando la formación de esferas celulares ya es muy notoria. A partir del día 1 tras la siembra, se apreciarán esferas celulares. Un análisis de la expresión de marcadores miogénicos por inmunofluorescencia de las células que conforman dichas esferas, debe detectar los marcadores Pax7 y MyoD1 , así como células MyHC+ (Figura 3). Esto es importante, dado que, las células precursoras miogénicas presentes en las esferas celulares se caracterizan porque son positivas para marcadores de célula satélite (Pax7) pero a su vez comienzan a expresar marcadores tempranos de diferenciación (MyoD1 ; (21 , 35)). La expresión de isoformas de miosina (marcadas por MyHC) en células precursoras en estado mononucleado es también indicativa del estado precursor miogénico de las mismas. Por lo tanto, en una realización preferida del primer o segundo aspecto de la invención, la composición que comprende células precursoras multipotentes aisladas del músculo cremáster humano del primer aspecto de la invención o la composición que comprende esferas obtenidas de cultivos de células aisladas del músculo cremáster del primer aspecto de la invención, se caracteriza porque dicha población celular es positiva para Pax7, MyoD1 y MyHC. 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. For this and as illustrated in the examples 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 ™ 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. 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. At this point, growth factors are added directly to the well at the indicated concentrations (Low serum growth supplement (LSGS; final concentration of 2% (1X), Rat recombinant basic fibroblast growth factor (rrFGF2; 80 ng / ml_ ), Rat recombinant epidermal growth factor (rrEGF; 40 ng / ml_). The first 3 days the medium is completely changed by centrifugation (5 minutes at 1500 rpm) to remove cell debris and select viable cells, and add the growth factors again every time, then these washes are done every 2 days and the culture is maintained until 7 or 9 days, when the formation of cell spheres is already very noticeable, from day 1 after planting, cell spheres will be appreciated An analysis of the expression of myogenic markers by immunofluorescence of the cells that make up said spheres, should detect the Pax7 and MyoD1 markers, as well as MyH cells C + (Figure 3) This is important, given that the myogenic precursor cells present in the cell spheres are characterized in that they are positive for satellite cell markers (Pax7) but in turn begin to express early differentiation markers (MyoD1; (21, 35)). 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.
Aún más preferiblemente, en las esferas obtenidas de cultivos de células aisladas del músculo cremáster del primer aspecto de la invención se observan los marcadores p75NTR y CD56 (Figura 4). Even more preferably, 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).
Es importante señalar que en esta memoria se entiende por “célula precursora miogénica”, a un conjunto probablemente heterogéneo de células que, en condiciones de cultivo específicas, alcanzan distintos estadios de diferenciación temprana siendo capaces de proliferar y mantener las capacidades de diferenciación a célula muscular. Estas células tendrían mejores capacidades de supervivencia, migración y toma en trasplantes a músculos dañados, en comparación con células más diferenciadas como los mioblastos. Es decir, la composición que comprende células precursoras multipotentes aisladas del músculo cremáster humano del primer aspecto de la invención así como la composición que comprende esferas obtenidas de cultivos de células aisladas del músculo cremáster del primer aspecto de la invención, se caracteriza por comprender dicho conjunto heterogéneo de células que, en condiciones de cultivo específicas, alcanzan distintos estadios de diferenciación temprana siendo capaces de proliferar y mantener las capacidades de diferenciación a célula muscular, denominado“célula precursora miogénica”. Por tanto, la composición del primer aspecto de la invención así como la composición del segundo aspecto de la invención son especialmente útiles para llevar a cabo trasplantes en músculos dañados. It is important to note that in this report it is understood as "myogenic precursor cell", a probably heterogeneous set of cells that, under specific culture conditions, reach different stages of early differentiation being able to proliferate and maintain differentiation capabilities to muscle cell . These cells would have better survival, migration and transplant capacities in damaged muscles, compared to more differentiated cells such as myoblasts. That is, 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. heterogeneous of cells that, under specific culture conditions, reach different stages of early differentiation being able to proliferate and maintain differentiation capabilities to a muscle cell, called "myogenic precursor cell". Therefore, the 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.
Por otro lado, en la presente invención se entiende por“marcador” a una proteína que distingue una célula (o grupo de células) de otra célula (o grupo de células). Por ejemplo, una proteína que se expresa en la superficie de células miogénicas pero no en otras células de una población celular actúa como proteína marcadora para las células precursoras miogénicas. “Positivo” significa que la célula expresa naturalmente el marcador. Para considerar que el marcador es expresado, debe estar presente a un“nivel detectable”. En esta memoria, por“nivel detectable” se entiende que el marcador puede detectarse por una de las metodologías estándar, tales como PCR, blotting, inmunofluorescencia o FACS. Se considera que un gen es expresado por una célula de la invención si puede ser razonablemente detectada tras 20 ciclos, preferiblemente 25 ciclos, y más preferiblemente 30 ciclos de PCR, que corresponde a un nivel de expresión en la célula de al menos 100 copias por célula. Se considera que un marcador no es expresado por una célula de la invención, si la expresión no puede ser detectada a un nivel de alrededor de 10 - 20 copias por célula. Entre estos niveles de positivo/negativo, la célula puede ser débilmente positiva para un determinado marcador. On the other hand, in the present invention "marker" is understood as a protein that distinguishes a cell (or group of cells) from another cell (or group of cells). For example, 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. It is considered that 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.
Por otro lado, la población celular de la invención es positiva para un determinado marcador si, al menos, el 20% de las células de la población muestra una expresión detectable del marcador, preferiblemente del 70%, 80%, 90%, 95%, y mucho más preferiblemente, del 98%. A veces, el 99% o el 100% de las células de la invención muestran una expresión detectable del marcador. Como en el caso de las células, la expresión puede ser detectada, por ejemplo pero sin limitarse, mediante técnicas de PCR, usando FACS (fluorescence activated cell sorting), o mediante inmunohistoquímica empleando anticuerpos específicos. On the other hand, 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. As in the case of cells, expression can be detected, for example but not limited, by PCR techniques, using FACS (fluorescence activated cell sorting), or by immunohistochemistry using specific antibodies.
El término“expresión natural”, o“expresa naturalmente” significa que las células no han sido manipuladas por tecnología recombinante, de ninguna forma, esto es, por ejemplo, que las células no han sido inducidas artificialmente a expresar estos marcadores o a modular la expresión de estos marcadores mediante la introducción en las células de material exógeno, como la introducción de promotores heterólogos, u otras secuencias unidas operativamente a cualquiera de los genes endógenos, o mediante la introducción de genes exógenos. The term "natural expression", or "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.
En una realización más preferida de este aspecto de la invención, la población de células precursoras miogénicas presentes en la composición del primer o segundo aspecto de la invención deriva de las células satélite del cremáster y mantiene las capacidades de célula madre gracias a su autorrenovación en el cultivo de mioesferas descrito en el segundo aspecto de la invención, equivalente a lo que se conoce como “célula satélite reserva” en los cultivos tradicionales de mioblastos. In a more preferred embodiment of this aspect of the invention, 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.
En esta memoria se entiende por célula“satélite del cremáster” a la célula madre procedente del músculo cremáster, que posee la capacidad de regenerar músculo a lo largo de la vida del individuo y diferenciarse en células precursoras miogénicas. In this report, 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.
Por otro lado, en la presente invención, se llevó a cabo la obtención de células miogénicas a partir de la formación de mioesferas presentes en el cultivo de proliferación del segundo aspecto de la invención. Para ello, se cambiaron las condiciones de cultivo para obtener una diferenciación miogénica (Figura 5). Las mioesferas se disgregaron con Tripsina-EDTA 0,25% durante 5 minutos a 37°C para después sembrar las células precursoras sobre una matriz extracelular (ECM) natural que favorece la adhesión y la diferenciación celular (66, 88), compuesta de extracto de membrana basal Cultrex® (2,77 mg/mL en concentración final), Netrina-4 (0,83 pg/mL), Netrina-G1a (0,83 pg/mL) y ácido hialurónico de bajo peso molecular (2,5 mg/mL) en PBS (pH 7,4). La solución de matriz extracelular se preparó teniendo en cuenta la concentración del extracto de membrana basal Cultrex® (Cultrex), dependiente del lote del producto. En base a este dato del volumen final se ajustaron los volúmenes del resto de los compuestos. En todos los casos se preparó una solución de matriz extracelular a una dilución 1 :3, preparando primero una mezcla para disolver los reactivos (equivalente al 1 de la dilución 1 :3) y después añadiendo el volumen necesario de PBS (pH 7,4) como diluyente a la mezcla para obtener la solución final (equivalente al 2 resultante del cálculo de la dilución 1 :3). En función del experimento a realizar se preparó una matriz extracelular con todos los reactivos mencionados o una matriz extracelular sin la presencia de Netrina-4 y Netrina-G1a, ajustando en este último caso el volumen de PBS (pH 7,4) que se utilizaría primero para disolver y mezclar los compuestos. Dado que los compuestos utilizados son termosensibles, con el fin de mantener su integridad y evitar una polimerización prematura, todos los reactivos se descongelaron gradualmente y se mantuvieron en hielo durante todo el proceso junto a la solución de matriz extracelular en preparación. De modo que tras reconstituir y descongelar todos los compuestos se preparó la solución de mezcla en un Falcon® de 50 mL que se mantuvo en hielo, y seguidamente se añadió el PBS (pH 7,4) como diluyente. Esta solución final fue agitada suavemente con cuidado de no formar burbujas para obtener una mezcla homogénea. A continuación, la matriz extracelular recién preparada se depositó en forma de gota (300 mI/pocillo) en placas estériles de 4 o 24 pocilios en los que previamente se habían colocado cubreobjetos autoclavados y se esparció inclinando la placa para formar una película uniforme. Una vez depositada la matriz, las placas se mantuvieron durante 24 horas a 37°C. Transcurrido este tiempo se retiró el exceso de líquido acumulado en los pocilios y se dejaron secar en la campana de flujo laminar durante 15 minutos para así obtener el sustrato deseado. Partiendo de las miosferas, una vez eliminado el medio utilizado para el cultivo en suspensión mediante una centrifugación de 5 minutos a 1500 rpm, las esferas agregadas en pellet fueron resuspendidas en 1 mL de solución de Tripsina-EDTA 0,25% y disgregadas manteniéndolas unos 5-7 minutos a 37°C para obtener una suspensión de células únicas con el objetivo de poder contarlas, depositarlas separadamente y conseguir una diferenciación más homogénea. La acción enzimática se detuvo diluyendo la solución con PBS (pH 7,2) y eliminándolo tras una centrifugación de 5 minutos a 1500 rpm para luego resuspender el pellet de células en 1 ml_ de medio para cultivo en adhesión y contar el número de células viables. Con esto se distribuyó la misma cantidad de células en cada pocilio, por lo general en placas de cultivo de 4 pocilios, poniendo 79.000 células por cm2 (150.000 células por pocilio); donde previamente se había depositado el sustrato que facilita la adhesión y promueve la diferenciación celular (ECM) y se había añadido medio para este cultivo hasta 500 pl. Estas células se mantuvieron así en la incubadora a 37°C y con 5% de C02, dejándolas reposar los primeros 2 días para después ir renovando la mitad del medio con medio fresco cada 2 días. Durante este cultivo de diferenciación se retiraron los factores de crecimiento y al medio de proliferación se le añadió un 10% de suero fetal bovino. En estas condiciones, al cabo de 7 días de cultivo, se obtuvo una diferenciación miogénica con generación de miotubos multinucleados. On the other hand, in the present invention, 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. To do this, 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). 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). Depending on the experiment to be performed, 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. This final solution was gently stirred with care not to form bubbles to obtain a homogeneous mixture. Next, 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. Starting from the myospheres, once the medium used for the suspension culture was removed by a 5 minute centrifugation at 1500 rpm, 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. With this, the same amount of cells was distributed in each well, usually in 4-well culture plates, placing 79,000 cells per cm2 (150,000 cells per well); where the substrate that facilitates adhesion and promotes cell differentiation (ECM) had previously been deposited and medium for this culture was added up to 500 pl. These cells were thus kept in the incubator at 37 ° C and with 5% C02, leaving them to rest for the first 2 days and then renew half of the medium with fresh medium every 2 days. During this differentiation culture, growth factors were removed and 10% fetal bovine serum was added to the proliferation medium. Under these conditions, after 7 days of culture, a myogenic differentiation was obtained with the generation of multinucleated myotubes.
Por lo tanto, un tercer aspecto de la invención se refiere a una composición que comprende una población celular de células miogénicas derivadas de la formación de las mioesferas presentes en el cultivo de proliferación del segundo aspecto de la invención o de una composición celular que comprende una población celular de células precursoras multipotentes derivadas o provenientes del músculo cremáster humano. Tal y como se muestra en los ejemplos de la presente invención, dicha composición celular de células miogénicas se caracteriza porque comprende una población celular positiva para los genes miogénicos Pax7, Myf5, MyoD1 , Myogenin, MyHC3 y MyHC2, en mayor cantidad comparada con un control negativo (tales como biopsias tomadas de la misma región pero no musculares). Se hace notar que la población celular referida, de ahora en adelante población celular de células miogénicas, debe comprender al menos una célula miogénica derivada o proveniente del músculo cremáster humano. En una realización preferida la población celular comprende al menos un 20%, preferiblemente un 40%, y aún más preferiblemente un 50%, 60%, 80%, 90%, 95%, o un 99% de células miogénicas derivadas o provenientes del músculo cremáster humano. Therefore, a third aspect of the invention relates to 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. As shown in the examples of the present invention, 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). It is noted that 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. In a preferred embodiment 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.
Por otro lado, también se pueden obtener células precursoras vasculogénicas o neurogénicas a partir de la formación de las mioesferas presentes en el cultivo de proliferación del segundo aspecto de la invención o a partir de una composición celular que comprende células precursoras multipotentes derivadas o provenientes del músculo cremáster humano. En este sentido, y con dicho propósito se hace notar que un cuarto aspecto de la invención se refiere a una composición que comprende una población celular de células precursoras vasculogénicas derivadas de la formación de las mioesferas presentes en el cultivo de proliferación del segundo aspecto de la invención o de una composición celular que comprende una población celular de células precursoras multipotentes derivadas o provenientes del músculo cremáster humano. Dicha composición celular se caracteriza porque comprende una población celular positiva para: p75NTR, SOX2, PAX3, DLK1 , RGS5, AOC3, ITGA7, ECRG4 y negativa para el marcador CD56 (Figura 4). Se hace notar que la población celular referida, de ahora en adelante población celular de células precursoras vasculogénicas, debe comprender al menos una célula precursora vasculogénica derivada o proveniente del músculo cremáster humano. En una realización preferida la población celular comprende al menos un 20%, preferiblemente un 40%, y aún más preferiblemente un 50%, 60%, 80%, 90%, 95%, o un 99% de células precursoras vasculogénicas derivadas o provenientes del músculo cremáster humano. On the other hand, 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. In this regard, and for that purpose it is noted that 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). It is noted that 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. In a preferred embodiment 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.
Adicionalmente, un quinto aspecto de la invención se refiere a una composición que comprende una población celular de células precursoras neurogénicas derivadas de la formación de las mioesferas presentes en el cultivo de proliferación del segundo aspecto de la invención o de una composición celular que comprende una población celular de células precursoras multipotentes derivadas o provenientes del músculo cremáster humano. Dicha composición celular se caracteriza porque comprende una población celular positiva para: p75NTR, SOX2, PAX3, CD56, SOX10, ERBB3, L1CAM, CDH2, CDH19, PMP22, PLP1 , DHH y negativa para el marcador SOX9 (Figura 4). Se hace notar que la población celular referida, de ahora en adelante población celular de células precursoras neurogénicas, debe comprender al menos una célula precursora neurogénica derivada o proveniente del músculo cremáster humano. En una realización preferida la población celular comprende al menos un 20%, preferiblemente un 40%, y aún más preferiblemente un 50%, 60%, 80%, 90%, 95%, o un 99% de células precursoras neurogénicas derivadas o provenientes del músculo cremáster humano. Additionally, a fifth aspect of the invention relates to 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). It is noted that 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. In a preferred embodiment 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.
Si se desea, las células presentes en cualquiera de las composiciones de la invención pueden ser modificadas genéticamente por cualquier método convencional incluyendo, a modo ilustrativo, no limitativo, procesos de transgénesis, deleciones o inserciones en su genoma que modifiquen la expresión de genes que sean importantes para sus propiedades básicas (proliferación, migración, diferenciación, etc.), o mediante la inserción de secuencias de nucleótidos que codifiquen proteínas de interés como, por ejemplo, proteínas con propiedades terapéuticas. Por tanto, en otra realización preferida, las células presentes en cualquiera de las composiciones de la invención han sido modificadas genéticamente. If desired, 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.
Por otro lado, si se desea, las células presentes en cualquiera de las composiciones de la invención pueden expandirse clonalmente usando un procedimiento adecuado para clonar poblaciones celulares. Por ejemplo, una población proliferada de células puede recogerse físicamente y sembrarse en una placa separada (o los pocilios de una placa“multi-pocillo”). Otra opción es que las células pueden subclonarse en una placa“multi-pocillo” en una relación estadística para facilitar la operación de colocar una única célula en cada pocilio (por ejemplo, desde aproximadamente 0,1 a cerca de una célula/pocillo o incluso de unas 0,25 a 0,5 células/pocillo, como por ejemplo 0,5 células/pocillo). Por supuesto, las células pueden clonarse a baja densidad (por ejemplo, en una placa de Petri u otro sustrato adecuado) y aislarlas de otras células usando dispositivos tales como anillos de clonación. La producción de una población clonal puede expandirse en cualquier medio de cultivo adecuado. En cualquier caso, las células aisladas pueden cultivarse hasta un punto adecuado cuando su fenotipo de desarrollo pueda evaluarse. On the other hand, if desired, the cells present in any of the compositions of the invention can be clonally expanded using a method suitable for cloning cell populations. For example, a proliferated population of cells can be physically collected and seeded on a separate plate (or the wells of a "multi-well" plate). Another option is that 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). Of course, 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. In any case, isolated cells can be cultured to a suitable point when their development phenotype can be evaluated.
En una realización más preferida de este aspecto, la población celular de cualquiera de los aspectos de la invención es una población aislada. In a more preferred embodiment of this aspect, the cell population of any aspect of the invention is an isolated population.
El término“aislada” indica que la célula o la población celular de la invención a la que se refiere, no se encuentran en su ambiente natural. Esto es, la célula o la población celular ha sido separada de su tejido circundante. Particularmente significa que dicha célula o la población celular está sustancialmente exenta (libre) de otras células normalmente presentes en el tejido, o en derivado del mismo, de un sujeto del que se haya extraído el músculo para el aislamiento de dicha célula o la población celular. En general, una célula está esencialmente libre de otras células presentes en el entorno de la misma cuando se separa de, al menos, el 60%, preferentemente de al menos el 80%, preferentemente de, al menos, el 90%, más preferentemente de, al menos, el 95%, aún más preferentemente de, al menos, el 96%, 97%, 98% o incluso 99%, de otras células presentes normalmente en dicho entorno. The term "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. . In general, 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.
También se refiere a las células o poblaciones celulares que han sido aisladas del organismo en el que se originan. El término también incluye células que han sido aisladas de un organismo y reintroducidas en el mismo organismo, o en otro. MÉTODO DE OBTENCIÓN DE CÉLULAS PRECURSORAS MULTIPOTENTES DE LA INVENCIÓN It also refers to cells or cell populations that have been isolated from the organism in which they originate. The term also includes cells that have been isolated from one organism and reintroduced into the same organism, or in another. METHOD OF OBTAINING MULTIPOTENT PRECURING CELLS OF THE INVENTION
En un sexto aspecto de la invención, la composición del segundo aspecto de la invención se obtiene por un método, de ahora en adelante método de obtención de células precursoras multipotentes de la invención, que comprende a) disgregar tejido biopsiado del músculo cremáster, b) separar las células intactas (viables) de los restos celulares c) cultivar las células del paso (b) en suspensión hasta la formación de esferas (5-8 días). In a sixth aspect of the invention, 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).
La disgregación del paso (a) puede llevarse a cabo por distintos métodos, entre ellos métodos mecánicos, métodos enzimáticos, o una combinación de ambos. Entre las enzimas empleadas se encuentran: acutasa, colagenasa (de diversos tipos, como A, I, II, IV, ó XI; o mezclas de las mismas), dispasa, DNasa I, elastasa, hialuronidasa, liberasa, papaína, pronasa, tripsina-EDTA, TrypLE, y muy habitualmente combinaciones de las anteriores. Esta lista se proporciona sólo a modo de ilustración, y no se pretende que sea limitante. Preferiblemente se emplea colagenasa tipo I o mezclas comerciales de distintas colagenasas como la liberasa. The disintegration of step (a) can be carried out by different methods, including mechanical methods, enzymatic methods, or a combination of both. Among 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. This list is provided by way of illustration only, and is not intended to be limiting. Preferably, type I collagenase or commercial mixtures of different collagenases such as liberase are used.
La separación del paso (b) se puede llevar a cabo por distintos métodos, entre ellos, pero sin limitarse a: filtración, decantación, centrifugación, métodos de separación magnéticos o basados en FACS, etcétera. 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.
El paso (c) se realiza en un medio de proliferación que no contiene suero y en condiciones de no adherencia al sustrato. Este medio al menos contiene: un medio de cultivo “base” como por ejemplo Neurobasal y factores de crecimiento de células precursoras que pueden incluir LSGS, FGF-2 y EGF, entre otros. Los días de cultivo entre pase y pase varían entre 4 y 8 días, dependiendo de diversos factores, y preferiblemente al menos 7 días para obtener esferas de tamaño adecuado para su manipulación posterior. 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.
El medio de cultivo“base” puede ser, preferiblemente pero sin limitarnos, un medio diseñado para el mantenimiento y la maduración a largo plazo de poblaciones de células neuronales prenatales y embrionarias puras, como el medio Neurobasal. Más preferiblemente el medio basal está suplementado con B27, y aún más preferiblemente con L-glutamina y una solución antibiótica. Entre el paso (b) y (c) puede haber, preferiblemente, un paso de centrifugación para obtener un precipitado celular. 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.
Las esferas de la composición del segundo aspecto de la invención se pueden seleccionar y disgregar para inyectarlas directamente, por ejemplo para el tratamiento de la incontinencia urinaria, se pueden utilizar directamente en terapia en forma de miosferas (sin disgregación celular) o se pueden diferenciar a mioblastos o células musculares estriadas para utilizar los constructos musculares como agentes terapéuticos o por su utilidad en el cribado de moléculas, biológicos u otros agentes terapéuticos cuya diana sea el músculo esquelético. En estos últimos casos sería preferible emplear las células con medios de soporte, como una matriz. 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.
En otra realización preferida de este aspecto, y con el objeto de proporcionar la composición del tercer aspecto de la invención, el método de obtención de células precursoras multipotentes de la invención además comprende: d) diferenciar las células precursoras multipotentes de las esferas del paso c) In another preferred embodiment of this aspect, and in order to provide the composition of the third aspect of the invention, the method of obtaining multipotent precursor cells of the invention further comprises: d) differentiating multipotent precursor cells from the spheres of step c )
Las células precursoras se pueden diferenciar en base a diversos protocolos existentes en la literatura, o bien de manera idéntica a como se diferencian los cultivos de mioblastos. Precursor cells can be differentiated based on various protocols existing in the literature, or identically to how myoblast cultures differ.
En otra realización preferida de este aspecto de la invención, la diferenciación del paso d) es a mioblastos. In another preferred embodiment of this aspect of the invention, the differentiation of step d) is to myoblasts.
En otra realización preferida de este aspecto de la invención, la diferenciación del paso d) es a miotubos. In another preferred embodiment of this aspect of the invention, the differentiation of step d) is to myotubes.
Es preferible emplear células precursoras multipotentes del cremáster que células satélite, ya que las células satélite son difíciles de cultivar y el número de células obtenido es pequeño, y además esos cultivos no presentan capacidad vasculogénica ni neurogénica. Las células precursoras miogénicas del cremáster tienen a su vez capacidad de proliferar y dar lugar a mioblastos, o de mantenerse como células precursoras en cultivo gracias a su capacidad de autorrenovación. 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.
COMPOSICIÓN FARMACÉUTICA DE LA INVENCIÓN PHARMACEUTICAL COMPOSITION OF THE INVENTION
Un séptimo aspecto de la invención se refiere a una composición tal y como se ha definido ésta en cualquiera de los aspectos o realizaciones reseñados con anterioridad, donde dicha composición .es una composición farmacéutica (de aquí en adelante“la composición farmacéutica de la presente invención”). Preferiblemente, la composición farmacéutica de la presente invención está referida a las composiciones de cualquiera de los aspectos primero, segundo o tercero de la invención. 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"). Preferably, the pharmaceutical composition of the present invention relates to the compositions of any of the first, second or third aspects of the invention.
Más preferiblemente la composición farmacéutica de la presente invención comprende una combinación de cualquiera de las composiciones de la invención. Aún más preferiblemente la composición farmacéutica comprende una combinación de la composición del tercer, cuarto y quinto aspecto de la invención. More preferably 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.
En otra realización preferida de este aspecto de la invención, la composición farmacéutica de la presente invención además comprende un vehículo farmacéuticamente aceptable. In another preferred embodiment of this aspect of the invention, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.
Las composiciones farmacéuticas de la presente invención se pueden utilizar en un método de tratamiento de manera aislada o junto con otros compuestos farmacéuticos. Por tanto, en otra realización más preferida de este aspecto de la invención, la composición farmacéutica de la presente invención además comprende otro principio activo. The pharmaceutical 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.
En este documento el término "componente activo", "sustancia activa", "sustancia farmacéuticamente activa", "componente activo" o "componente farmacéuticamente activo" significa cualquier componente que potencialmente proporciona una actividad farmacológica u otro efecto diferente en el diagnóstico, la curación, la mitigación, el tratamiento, o prevención de una enfermedad, o que afecte a la estructura o función del cuerpo humano u otros animales. Ejemplos de componentes activos de origen biológico incluyen a factores de crecimiento, hormonas y citoquinas. En el estado del arte se conocen una variedad de agentes terapéuticos y podrían ser identificados por sus efectos. Determinados agentes terapéuticos son capaces de regular la proliferación y la diferenciación celular. Como ejemplo se incluyen nucleótidos, fármacos quimioterapéuticos, hormonas, proteínas no específicas (que no son anticuerpos), oligonucleótidos (por ejemplo, oligonucleótidos anti sentido que se unen a una secuencia de ácido nucleído diana (por ejemplo, secuencia de ARN), péptidos y peptidomiméticos. También podrían actuar como componente activo otras células. In this document the term "active component", "active substance", "pharmaceutically active substance", "active component" or "pharmaceutically 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. Examples of 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. 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.
En otra realización preferida de este aspecto, la composición farmacéutica de la presente invención comprende, adicionalmente, uno o varios excipientes farmacéuticamente aceptables. El término "excipiente farmacéuticamente aceptable" tal como se utiliza aquí se refiere al hecho de que debe ser aprobado por una agencia reguladora de un gobierno federal o un gobierno nacional o uno de los enumerados en la Farmacopea de Estados Unidos o de la Farmacopea Europea, o alguna otra farmacopea reconocida generalmente para su uso en animales y en los seres humanos. El término "vehículo" se refiere a un diluyente, excipiente, vehículo o adyuvante con el que las células madre, células progenitoras o células diferenciadas de la invención, las células de la invención, así como las células de la población de células de la invención, deben ser administradas; obviamente, dicho vehículo debe ser compatible con las células. Ejemplos ilustrativos, dicho vehículo incluyen cualquier vehículo fisiológicamente compatible, por ejemplo, soluciones isotónicas (por ejemplo, solución salina estéril (0,9% NaCI), solución tampón fosfato salino (PBS), solución de Ringer-lactato, etc), complementados opcionalmente con suero, preferentemente con suero autólogo; medios de cultivo (por ejemplo, DMEM, RPMI, McCoy, etc), o, preferentemente, un medio sólido, semisólido, gelatinoso o viscoso apoyo, tales como el colágeno, colágeno-glicosamino-glicano, fibrina, cloruro de polivinilo, ácidos poli-aminoácidos, tales como polilisina, o poliornitina, hidrogeles, agarosa, sulfato de dextrano de silicona. Por otra parte, si se desea, el medio de soporte puede en realizaciones especiales, contener, factores de crecimiento u otros agentes. Si el soporte es sólido, semisólido, o gelatinoso, las células pueden introducirse en la fase líquida de un vehículo que se trata posteriormente de manera que se convierte en una fase más sólida. En alguna realización de la invención en el que el vehículo tiene una estructura sólida, dicho vehículo se puede configurar de acuerdo con la forma de la lesión, como por ejemplo en forma de anillo para el tratamiento de la incontinencia urinaria debido a daño en el músculo estriado del esfínter urinario externo. In another preferred embodiment of this aspect, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" as used herein 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. The term "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. Illustrative examples, 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. On the other hand, if desired, the support medium may in special embodiments, contain, growth factors or other agents. If the support is solid, semi-solid, or gelatinous, the cells can be introduced into the liquid phase of a vehicle that is subsequently treated so that it becomes a more solid phase. In some embodiment of the invention 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.
La composición farmacéutica de la invención puede, si se desea, contener también, cuando sea necesario, aditivos para aumentar y/o controlar el efecto terapéutico deseado de las células, por ejemplo agentes de tamponamiento, agentes de superficie activos, conservantes, etc. El vehículo farmacéuticamente aceptable puede comprender un medio de cultivo celular que mantenga la viabilidad de las células. El medio estará generalmente libre de suero con el fin de evitar provocar una respuesta inmune en el receptor. El transportista será generalmente tampón y/o libre de pirógenos. Además, para la estabilización de la suspensión celular, es posible añadir agentes quelantes de metales. La estabilidad de las células en el medio líquido de la composición farmacéutica de la invención puede ser mejorada mediante la adición de sustancias adicionales, tales como, por ejemplo, ácido aspártico, ácido glutámico, etc. Dichas sustancias farmacéuticamente aceptables que pueden ser utilizadas en la composición farmacéutica de la invención son generalmente conocidas por un experto en la técnica y normalmente se utilizan en la producción de composiciones celulares. Los ejemplos de vehículos farmacéuticos adecuados se describen en "Flemington’s Pharmaceutical Sciences " por E.W. Martin. Se puede encontrar información adicional sobre dichos vehículos en cualquier manual de la tecnología farmacéutica (es decir, de farmacia galénica). 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. In addition, 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. Examples of 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).
La composición farmacéutica de la invención se puede administrar en una forma farmacéutica de administración adecuada. Por lo tanto, la composición farmacéutica de la invención se formulará de acuerdo con la forma de administración elegida. La formulación se adaptará a la forma de administración. En una realización especial, la composición farmacéutica se prepara en una forma de dosificación líquida, sólida o semisólida, por ejemplo, en forma de suspensión, con el fin de ser administrada mediante la implantación, inyección o infusión al sujeto que necesita tratamiento. Como ejemplo, de manera ilustrativa y no limitativa, se presenta una posible formulación de la composición farmacéutica de la invención en una suspensión estéril con un excipiente farmacéuticamente aceptable, por ejemplo, una solución isotónica, por ejemplo, solución tampón fosfato salino (PBS), o cualquier otro vehículo adecuado, farmacéuticamente aceptable, para la administración a un sujeto por vía parenteral, aunque otras vías de administración también pueden ser utilizadas. 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. In a special embodiment, 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. As an example, in an illustrative and non-limiting manner, 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.
La administración de la composición farmacéutica de la invención al sujeto que lo necesite se llevará a cabo utilizando medios convencionales. En una realización particular, la composición farmacéutica de la invención puede ser administrada al sujeto por vía parenteral mediante dispositivos adecuados, tales como jeringas, catéteres, trocares, cánulas, etc. En todos los casos, la composición farmacéutica de la invención se puede administrar usando equipos, aparatos y los dispositivos adecuados para la administración de composiciones celulares y conocidas por una persona experta en la técnica. En otra realización, la administración directa de la composición farmacéutica de la invención para el sitio que se pretende beneficiar podría ser ventajosa. En este método, la administración directa de la composición farmacéutica de la invención para el órgano o tejido deseado se puede lograr mediante la administración directa (por ejemplo, por inyección, etc) en la superficie externa del órgano o tejido afectado por la inserción de un dispositivo adecuado, por ejemplo, una cánula adecuada, por infusión (incluyendo mecanismos de flujo inverso) o por otros medios descritos en esta patente o conocidos en el estado del arte. La composición farmacéutica de la invención puede almacenarse hasta el momento de su aplicación por los métodos convencionales conocidos por el experto en la materia. Para el almacenamiento a corto plazo (menos de 6 horas), la composición farmacéutica de la invención puede ser almacenada a temperatura ambiente o inferior, en un recipiente sellado, suplementado o no con una solución de nutrientes. Para almacenamiento a medio plazo (menos de 48 horas) se lleva a cabo preferiblemente entre 2-8°C, y en la composición farmacéutica de la invención se incluye, además, una solución isosmótica tamponada en un recipiente que esté hecho o recubierto con un material que evite la adhesión celular. El almacenamiento a más largo plazo se lleva a cabo preferiblemente por criopreservación y almacenado en condiciones que promuevan la conservación de la función celular. The administration of the pharmaceutical composition of the invention to the subject in need will be carried out using conventional means. In a particular embodiment, the pharmaceutical composition of the invention can be administered to the subject parenterally by suitable devices, such as syringes, catheters, trocars, cannulas, etc. In all cases, 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. In another embodiment, direct administration of the pharmaceutical composition of the invention to the site that is intended to benefit could be advantageous. In this method, 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. The pharmaceutical composition of the invention can be stored until its application by conventional methods known to those skilled in the art. For short-term storage (less than 6 hours), 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. For 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.
En una realización concreta, la composición farmacéutica de la invención se puede utilizar en terapia combinada. Estos medicamentos adicionales podrían formar parte de la misma composición farmacéutica o, alternativamente, ser suministrado en forma de una composición separada para su administración simultánea o sucesiva (secuencial en el tiempo) a la administración de la composición farmacéutica de la invención. In a specific embodiment, the pharmaceutical composition of the invention can be used in combination therapy. These 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.
DISPOSITIVO MÉDICO Y TEJIDOS QUE COMPRENDEN CÉLULAS PRECURSORAS MULTIPOTENTES MEDICAL DEVICE AND FABRICS THAT INCLUDE MULTIPOTENT PRECURSOR CELLS
En una realización adicional, las composiciones de la invención (cualquiera de las composiciones descritas en los aspectos primero a quinto de la presente invención) pueden ser implantadas o inyectadas en el paciente junto con un componente material de apoyo. Esto podría asegurar que las células permanezcan en el lugar adecuado dentro del paciente. En otra realización preferida, el material de soporte es de origen natural o sintético. En una realización más preferida, el material de soporte de origen natural se selecciona de la lista que consiste en: seda, membranas serosas mesentéricos bovinas descelularizadas, pericardio bovino descelularizado y combinaciones de los mismos. En una realización aún más preferida, el material de soporte es hilo con una estructura de monofilamento o multifilamento, y en una realización particular, el material de soporte es una lámina de nanofibras de seda. In a further embodiment, the compositions of the invention (any of the compositions described in the first to fifth aspects of the present 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. In another preferred embodiment, the support material is of natural or synthetic origin. In a more preferred embodiment, 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. In an even more preferred embodiment, the support material is thread with a monofilament or multifilament structure, and in a particular embodiment, the support material is a silk nanofiber sheet.
Otros ejemplos de material de soporte incluyen como base el colágeno, fibrina, laminina, fibronectina y un material artificial, o combinaciones de los mismos. Esta lista se proporciona sólo a modo de ilustración, y no se pretende que sea limitante. Para el experto en la técnica sería evidente utilizar cualquier combinación de uno o más componentes para formar la matriz. En una realización adicional, las células de las composiciones de la invención pueden estar contenidas dentro de una microesfera. Dentro de esta realización, las células pueden encapsularse dentro del centro de la microesfera. También dentro de esta realización, las células pueden estar incrustadas en el material de la matriz de la microesfera. El material de la matriz puede incluir cualquier polímero biodegradable adecuado, incluyendo pero no limitado a, alginatos, polietilenglicol (PLGA), fibrina y sericina y poliuretanos, o cualquiera de sus combinaciones. Esta lista se proporciona sólo a modo de ejemplo, y no se pretende que sea limitante. Other examples of support material 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. In a further embodiment, 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.
En una realización adicional, las células de las composiciones de la invención pueden adherirse a un dispositivo médico, de ahora en adelante dispositivo médico de la invención, destinado a la implantación. Ejemplos de dispositivos médicos incluyen los stents, alfileres, puntos de sutura, fracturas, marcapasos, prótesis articulares, piel artificial, y varillas. Esta lista se proporciona sólo a modo de ilustración, y no se pretende que sea limitante. Para el experto en la técnica sería evidente adherir las células al dispositivo médico por distintos métodos. Por ejemplo, las células pueden adherirse al dispositivo médico utilizando fibrina, uno o más miembros de la familia de las integrinas, uno o más miembros de la familia de las cadherinas, uno o más miembros de la familia de las selectinas, una o más moléculas de adhesión celular (CAMs), uno o más miembros de la familia de las inmunoglobulinas y uno o más adherentes artificiales. Esta lista se proporciona sólo a modo de ilustración, y no se pretende que sea limitante. Para el experto en la técnica sería evidente utilizar cualquier combinación de uno o más componentes adherentes. In a further embodiment, the cells of the compositions of the invention may adhere to a medical device, hereinafter medical device of the invention, intended for implantation. Examples of 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. For example, 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. 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 adherent components.
Por tanto, un octavo aspecto de la invención se refiere a un dispositivo médico, de ahora en adelanta dispositivo médico de la invención, que comprende las células de las composiciones de la invención. Thus, 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.
Un noveno aspecto de la invención se refiere a un método in vitro para la preparación de un tejido artificial, de ahora en adelante tejido artificial de la invención, que comprende: a) sembrar en un material de soporte las células de cualquiera de las composiciones de la invención , y b) cultivar dichas células en el material de soporte de (a) en un medio de cultivo apropiado. 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.
Otro aspecto de la invención se refiere al tejido artificial de la invención obtenido por el método in vitro anteriormente descrito. Que el medio de cultivo sea adecuado para la obtención del tejido artificial es conocido en el estado de la técnica. Por ejemplo, en el caso de las células derivadas del músculo cremáster que constituyen una población total de células debería contener por ejemplo medio Neurobasal A, suplementos (B27 y L-Glutamina) y factores de crecimiento como FGF, EGF, LSGS. 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. For example, 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.
"Soporte" tal como se utiliza aquí, se refiere a cualquier dispositivo o material que puede servir como una base o matriz para el crecimiento de las células derivadas del músculo cremáster que constituyen una población total de células precursoras multipotentes de la invención, y más preferiblemente para el crecimiento y diferenciación de las células precursoras multipotentes de la invención. "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.
En otra realización preferida, el material de soporte es de origen natural o sintético. En una realización más preferida, el material de soporte de origen natural se selecciona de la lista que consiste en: seda, membranas serosas bovinas descelularizadas aisladas del mesenterio, pericardio bovino descelularizado y combinaciones de los mismos. En una realización aún más preferida, el material de soporte es hilo con una estructura de monofilamento o multifilamento, y en una realización particular, el material de soporte es una lámina de nanofibras de seda. In another preferred embodiment, the support material is of natural or synthetic origin. In a more preferred embodiment, 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. In an even more preferred embodiment, the support material is thread with a monofilament or multifilament structure, and in a particular embodiment, the support material is a silk nanofiber sheet.
Otros ejemplos de material de soporte incluyen como base el colágeno, fibrina, laminina, fibronectina, uno o varios polisacáridos, y un material artificial, o cualquiera de sus combinaciones. Esta lista se proporciona sólo a modo de ilustración, y no se pretende que sea limitante. Para el experto en la técnica sería evidente utilizar cualquier biomaterial, biomaterial ortopédico convencional o avanzada o una combinación de biomateriales. Other examples of support material 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.
En una realización más preferida, el material de soporte es hilo con estructura de monofilamento o multifilamento. In a more preferred embodiment, the support material is thread with monofilament or multifilament structure.
Uno de los mayores inconvenientes de la sutura de tejidos, es el hecho de que el diámetro de la aguja es mayor que el hilo, de modo que el punto de la aguja de inserción no será totalmente ocupado por este último, la generación de las zonas a través del cuales puede ocurrir la pérdida de fluidos. Este cierre deficiente de las heridas está con frecuencia asociado a complicaciones postoperatorias, tales como las anastomosis intestinales realizadas en pacientes con carcinoma o diverticulosis, que se realizan debido a una resección por una enfermedad intestinal, uniéndose posteriormente los dos extremos sanos. En estos pacientes, debido a un cierre incompleto, se podrían perder las heces e invadir los tejidos circundantes, lo que provocaría peritonitis, con el consiguiente riesgo para la vida del paciente. Este riesgo se incrementa en pacientes con un grosor reducido de la pared intestinal, como en el caso de una enfermedad inflamatoria del intestino. Las células precursoras multipotentes se pueden aplicar en la sutura de manera que la abertura se sella generada por el paso del hilo a través de la costura. Por lo tanto, en otra realización preferida, el material de soporte es un hilo de sutura unido a una aguja. 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. In these patients, due to incomplete closure, 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.
El término "sutura" se refiere a un hilo o fibra u otro material de cierre que se puede utilizar para coser una herida. The term "suture" refers to a thread or fiber or other closure material that can be used to sew a wound.
El uso de grapas es una alternativa al método clásico de sutura. Permite el cierre primario de tejido en menos tiempo, reducir la pérdida de sangre, reducir la contaminación y preservar el flujo sanguíneo. Un factor limitante en el uso de grapas como un método de curación de atención primaria es la posibilidad de tener acceso a la parte superior e inferior del tejido a unir. También, debido a la fuerza ejercida la inserción de las grapas puede causar desgarro del tejido. Luego, en otra realización preferida, las grapas son el material de soporte. The use of staples is 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.
USOS MÉDICOS DE LA INVENCIÓN MEDICAL USES OF THE INVENTION
Otro aspecto de la invención se refiere al uso de a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, o c) el tejido artificial de la invención, en la elaboración de un medicamento, o alternativamente, se refiere a a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, c) el tejido artificial de la invención, para su uso en medicina o terapia. 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.
Otro aspecto de la invención se refiere al uso de a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, c) el tejido artificial de la invención, en la elaboración de un medicamento para aumentar parcial o completamente, restaurar o reemplazar la actividad funcional de un tejido u órgano enfermo o dañado, o alternativamente, se refiere a a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, c) el tejido artificial de la invención, para aumentar parcial o completamente, restaurar o reemplazar la actividad funcional de un tejido u órgano enfermo o dañado. 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.
En una realización preferida de este aspecto de la invención, el tejido u órgano enfermo o dañado es el esfínter uretral, el esfínter anal, o una combinación de ambos. In a preferred embodiment of this aspect of the invention, the diseased or damaged tissue or organ is the urethral sphincter, the anal sphincter, or a combination of both.
Por tanto, otro aspecto de la invención se refiere al uso de a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, c) el tejido artificial de la invención, en la elaboración de un medicamento para el tratamiento de la incontinencia urinaria, la incontinencia fecal, o ambas, o alternativamente, se refiere a a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, c) el tejido artificial de la invención, para el tratamiento de la incontinencia urinaria, la incontinencia fecal, o ambas. Otro aspecto de la invención se refiere al uso de a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, c) el tejido artificial de la invención, en la elaboración de un medicamento para la regeneración del músculo estriado, o alternativamente, se refiere a a) cualquiera de las composiciones de la invención, preferiblemente la composición del primer, segundo o tercer aspecto de la invención, b) el dispositivo médico de la invención, c) el tejido artificial de la invención, para la regeneración del músculo estriado. Therefore, 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.
A lo largo de la descripción y de las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, suplementos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración y no pretenden limitar la presente invención. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, supplements, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to limit the present invention.
EJEMPLOS DE LA INVENCIÓN EXAMPLES OF THE INVENTION
1.- Caracterización histológica del músculo cremáster masculino 1.- Histological characterization of the male cremaster muscle
Se obtuvieron biopsias del músculo cremáster tras aprobación ética y consentimiento informado de pacientes de varicocele o quistes de cordón espermático. Para los análisis histológicos (Figura 1), las biopsias se seccionaron en cortes seriados de 7-10 mieras de grosor. La expresión de distintas proteínas marcadoras del músculo esquelético se detectó a través de inmunofluorescencia. Como primer análisis, se realizó una tinción de hematoxilina-eosina donde claramente se observaron los paquetes de fibras musculares y se cuantificó el número de fibras que presentaban núcleos centrales, siendo este del 0.8%. El cremáster está mayoritariamente compuesto por fibras de tipo I (lentas), aunque contiene en torno a un 10% de fibras rápidas. La detección del marcador específico de células satélite Pax7 demostró la existencia de un 2% de células Pax7+ in situ. Crema muscle biopsies were obtained after ethical approval and informed consent of patients with varicocele or spermatic cord cysts. For histological analyzes (Figure 1), the biopsies were sectioned in serial sections 7-10 microns thick. The expression of different skeletal muscle marker proteins was detected through immunofluorescence. As a first analysis, hematoxylin-eosin staining was performed where muscle fiber bundles were clearly observed and the number of fibers that had central nuclei was quantified, this being 0.8%. The cremaster is mostly composed of type I (slow) fibers, although it contains about 10% of fast fibers. The detection of the specific marker of Pax7 satellite cells demonstrated the existence of 2% Pax7 + cells in situ.
2.- Obtención y cultivo de células precursoras multipotentes a partir de biopsias del músculo cremáster a) Distintos tipos de células precursoras presentes en el cultivo de esferas. 2.- Obtaining and cultivating multipotent precursor cells from cream muscle biopsies a) Different types of precursor cells present in the culture of spheres.
A modo de ejemplo, la Figura 2 muestra las esferas obtenidas de cultivos de células obtenidas de dermis de ratón, y cómo esas dermoesferas contienen células precursoras multipotentes de diversos tipos, incluyendo células precursoras miogénicas, células precursoras vasculogénicas y células precursoras neurogénicas. b) Procesado del músculo cremáster para el cultivo celular By way of example, 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
La biopsia de músculo cremáster (2 cm2) se transportó en frío en 50 mi de medio HBSS con un 1 % de Fungizona y un 1 % de Penicilina. La biopsia se agitó para la oxigenación del músculo. En cabina de cultivos, se lavó la biopsia en HBSS frío y traspasó con las pinzas a la placa p100 donde se troceó en fragmentos pequeños con unas pinzas esterilizadas y un bisturí quirúrgico. Los fragmentos resultantes se traspasaron a un tubo estéril con unos 15 mi de Colagenasa tipo I filtrada y se incubaron a 37°C y en agitación a 180 rpm durante 1-2h aproximadamente. 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.
El tejido disgregado en colagenasa se pasó a un tubo estéril de 50 mi al que se añadieron 20 mi de medio de proliferación para inactivar la colagenasa. Posteriormente se filtró la solución celular con un Cell Strainer de 40 pm. Para retirar la colagenasa se centrifugó la solución a 1500 rpm 5 min a 23°C. El pellet resultante que contenía las células precursoras multipotentes del músculo cremáster se cultivó y se analizó mediante diferentes técnicas. c) Cultivo celular para la formación de esferas 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
Para el cultivo celular se resuspendió el pellet en medio de cultivo para condiciones no adherentes, llamado medio de proliferación (medio Neurobasal™ A suplementado con 2% de suplemento B27 50X, 1% de L-glutamina 200 mM y 1% de solución de penicilina/estreptomicina 100X) y se sembraron en placas no tratadas. Tras añadir las células al pocilio se añadieron los factores de crecimiento (LSGS, FGF2, EGF). El medio de cultivo se cambió durante todos los días durante los primeros 3 días, para después hacerlo en días alternos hasta los 7 días. A partir del día 1 tras la siembra se pudieron apreciar esferas celulares. Los lavados se hicieron recogiendo las células de cada pocilio en un tubo estéril de 50 mi y centrifugando a 1500 rpm durante 5 min a RT. El pellet se resuspendió de nuevo en 1 mi de medio de proliferación y se sembraron las células en un nuevo pocilio con medio y factores de crecimiento. En los casos en los que hubo bastante densidad celular, se expandieron las células en dos pocilios. For the cell culture, the pellet was resuspended in culture medium for non-adherent conditions, called proliferation medium (Neurobasal ™ 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. After adding the cells to the well, growth factors (LSGS, FGF2, EGF) were added. 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 and growth factors. In cases where there was enough cell density, the cells were expanded in two wells.
A día 0 y 7 de cultivo, se realizó un análisis de la expresión de marcadores miogénicos por inmunofluorescencia. Se detectó tinción para los marcadores de células satélite Pax7 y MyoD1 a día 7, así como células MyHC+ (Figura 3). Así mismo, se detectó tinción para los marcadores de células precursoras neurogénicas y vasculogénicas p75NTR y CD56 (Figura 4). d) Cultivo celular para la diferenciación miogénica On day 0 and 7 of culture, an analysis of the expression of myogenic markers by immunofluorescence was performed. Staining was detected for the Pax7 and MyoD1 satellite cell markers at day 7, as well as MyHC + cells (Figure 3). Likewise, staining was detected for the markers of neurogenic and vasculogenic precursor cells p75NTR and CD56 (Figure 4). d) Cell culture for myogenic differentiation
Tras la formación de las mioesferas en cultivo de proliferación, se cambiaron las condiciones de cultivo para obtener una diferenciación miogénica (Figura 5). Las mioesferas se disgregaron con Tripsina-EDTA 0,25% durante 5 minutos a 37°C para después sembrar las células precursoras sobre una matriz extracelular (ECM) natural que favorece la adhesión y la diferenciación celular (66, 88), compuesta de extracto de membrana basal Cultrex® (2,77 mg/mL en concentración final), Netrina-4 (0,83 pg/mL), Netrina-G1a (0,83 pg/mL) y ácido hialurónico de bajo peso molecular (2,5 mg/mL) en PBS (pH 7,4). Durante este cultivo de diferenciación se retiraron los factores de crecimiento y al medio de proliferación se le añadió un 10% de suero fetal bovino. En estas condiciones, al cabo de 7 días de cultivo, se obtuvo una diferenciación miogénica con generación de miotubos multinucleados. After the formation of the myospheres in proliferation culture, the culture conditions were changed 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). During this differentiation culture, growth factors were removed and 10% fetal bovine serum was added to the proliferation medium. Under these conditions, after 7 days of culture, a myogenic differentiation was obtained with the generation of multinucleated myotubes.
Se observó que los miotubos expresaban proteínas específicas de músculo estriado como la MyHC y presentaban las estriaciones características de este tipo muscular. Además, también se observaron células precursoras PAX7 positivas, posiblemente células satélite reserva. It was observed that the myotubes expressed specific striated muscle proteins such as MyHC and presented the characteristic striations of this muscular type. In addition, PAX7 positive precursor cells, possibly reserve satellite cells, were also observed.
Para realizar el análisis de la expresión de genes determinantes para la miogénesis mediante retrotranscripción seguida de PCR cuantitativa a tiempo real (RT-qPCR), el ARNm de las muestras se extrajo con la ayuda del kit miRNeasy Mini Kit en el equipo QIAcube (Qiagen). Para la preparación de las muestras antes de la extracción de ARNm, estas se pasaron a tubos Eppendorf® Safe-Lock Biopur® RNase free y se añadió Qiazol® en un volumen final de 700 pl_ para guardarlas a -80°C durante un mínimo de 2 horas. A la hora de hacer la extracción de ARNm, se descongelaron, se añadió 140 mI_ de cloroformo a cada muestra, se agitaron, se realizó un vortex de 15 segundos y se dejaron reposar 2 o 3 minutos a temperatura ambiente. Después, se centrifugaron a 12000 g durante 15 minutos a 4°C. Se recogió la fase acuosa de las tres fases que se diferencian tras la centrifugación para pasarla a tubos estériles que se colocaron en la maquina QIAcube junto al resto de materiales y reactivos necesarios. Se procedió a la extracción automatizada de ARNm para finalmente obtener 30 mI_ de las muestras definitivas. Se midió la cantidad y pureza del ARNm obtenido en el NanoDrop™ ND-1000 Spectrophotometer (ThermoFisher). Seguidamente, el ARNm extraído se convirtió en ADN complementario (ADNc) mediante una PCR de transcripción reversa (RT-PCR). Para ello se utilizó el kit de transcripción reversa de ADN complementario de alta capacidad. Se retrotranscribieron 1 ,25 pg de ARNm de cada muestra, mezclándolo con los diferentes componentes del kit en un volumen final de 50 mI_ y se realizó la PCR. La RT-qPCR del ADNc de las muestras se realizó en la máquina 7900 HT Fast Real-Time PCR System (Applied Biosystems) y se utilizaron sondas TaqMan® de expresión génica (0,5 pL por pocilio) en solución TaqMan® Gene Expression Master Mix (5 pL por pocilio). Se añadieron 20,2 ng de ADNc en agua RNase-Free en un volumen final de 4,5 pL por pocilio. De esta manera, el volumen final de reacción fue de 10 pL por pocilio, cada muestra se amplificó por triplicado en la placa de ensayo). To perform the analysis of gene expression determinants for myogenesis by retrotranscription followed by real-time quantitative PCR (RT-qPCR), the mRNA from the samples was extracted with the help of the miRNeasy Mini Kit in the kit QIAcube (Qiagen). For sample preparation before mRNA extraction, these were transferred to Eppendorf® Safe-Lock Biopur® RNase free tubes and Qiazol® was added in a final volume of 700 pl_ to store at -80 ° C for a minimum of 2 hours. At the time of mRNA extraction, they were thawed, 140 ml of chloroform was added to each sample, stirred, a 15-second vortex was performed and allowed to stand for 2 or 3 minutes at room temperature. Then, they were centrifuged at 12000 g for 15 minutes at 4 ° C. The aqueous phase was collected from the three phases that are differentiated after centrifugation to pass it to sterile tubes that were placed in the QIAcube machine along with the rest of the necessary materials and reagents. Automated mRNA was extracted to finally obtain 30 ml of the final samples. The quantity and purity of the mRNA obtained in the NanoDrop ™ ND-1000 Spectrophotometer (ThermoFisher) was measured. Next, the extracted mRNA was converted to complementary DNA (cDNA) by reverse transcription PCR (RT-PCR). For this, the high capacity complementary DNA reverse transcription kit was used. 1.25 pg of mRNA from each sample was re-transcribed, mixing it with the different kit components in a final volume of 50 mI_ and PCR was performed. The RT-qPCR of the cDNA of the samples was performed on the 7900 HT Fast Real-Time PCR System (Applied Biosystems) and TaqMan® gene expression probes (0.5 pL per well) were used in TaqMan® Gene Expression Master solution Mix (5 pL per well). 20.2 ng cDNA in RNase-Free water was added in a final volume of 4.5 pL per well. Thus, the final reaction volume was 10 pL per well, each sample was amplified in triplicate on the test plate).
Se analizaron los resultados relativos a la expresión del gen endógeno Tbp, donde se observó cierta variabilidad en la expresión entre las muestras, pero todas ellas expresaban los genes miogénicos Pax7, Myf5, MyoD1 , Myogenin, MyHC3 y MyHC2, y en mayor cantidad comparada con el control negativo. e) Regeneración muscular in vivo The results related to the expression of the endogenous Tbp gene were analyzed, where some variability in the expression between the samples was observed, but all of them expressed the myogenic genes Pax7, Myf5, MyoD1, Myogenin, MyHC3 and MyHC2, and in greater quantity compared to The negative control. e) Muscle regeneration in vivo
Se llevó a cabo un ensayo in vivo para evaluar la capacidad regenerativa de estas células frente a un daño agudo en el músculo tibial anterior (TA) del ratón (Figura 6). Se siguió un diseño experimental en el que se producía el daño muscular por inyección de cardiotoxina (CTX) en las dos extremidades posteriores de 6 ratones inmunodeficientes NSG (NOD scid gamma). Al día siguiente, se inyectaron las células precursoras miogénicas de cremáster sólo en una de las extremidades dañadas, y se analizaron los resultados a las 4 semanas de la inyección celular. Al término del experimento se extrajeron los músculos TA de las dos extremidades, se congelaron en isopentano enfriado en nitrógeno líquido para posteriormente realizar cortes seriados del músculo completo. Estos cortes seriados fueron analizados mediante inmunofluorescencia utilizando los anticuerpos anti-Pax7, y anti-hl_aminA/C y hDystrophin (específicos de músculo humano, que no reaccionan con el músculo del ratón) para detectar las células humanas y su posible contribución a las fibras regeneradas. Se detectaron células humanas por la expresión de la proteína Lamín A/C y hDystrophin en todas las muestras analizadas, donde se observó una correlación entre el número de estas células y el número de estas fibras generadas en cada muestra. A su vez se realizó una cuantificación del número de células humanas Lamín A/C positivas que co-expresaban el marcador de las células satélite Pax7, obteniéndose aproximadamente un 2%. An in vivo assay was carried out to assess the regenerative capacity of these cells against acute damage to the anterior tibial muscle (TA) of the mouse (Figure 6). An experimental design was followed in which muscle damage was caused by cardiotoxin injection (CTX) in the two hind limbs of 6 NSG immunodeficient mice (NOD scid gamma). The next day, the myogenic crematerial precursor cells were injected into only one of the damaged limbs, and the results were analyzed 4 weeks after the cell injection. At the end of The TA muscles were extracted from both extremities, frozen in liquid nitrogen-cooled isopentane to subsequently make serial cuts of the entire muscle. These serial sections were analyzed by immunofluorescence using the anti-Pax7, and anti-hl_aminA / C and hDystrophin antibodies (specific to human muscle, which do not react with the mouse muscle) to detect human cells and their possible contribution to regenerated fibers . Human cells were detected by the expression of the Lamin A / C protein and hDystrophin in all the samples analyzed, where a correlation was observed between the number of these cells and the number of these fibers generated in each sample. In turn, a quantification of the number of human Lamin A / C positive cells that co-expressed the Pax7 satellite cell marker was performed, obtaining approximately 2%.
REFERENCIAS REFERENCES
1. Barker N, Bartfeld S, Clevers H. Tissue-resident adult stem cell populations of rapidly self-renewing organs. Cell Stem Cell. 2010;7(6):656-70. 1. Barker N, Bartfeld S, Clevers H. Tissue-resident adult stem cell populations of rapidly self-renewing organs. Cell Stem Cell. 2010; 7 (6): 656-70.
2. Mauro A. Satellite cell of skeletal muscle fibers. The Journal of biophysical and biochemical cytology. 1961 ;9:493-5.  2. Mauro A. Satellite cell of skeletal muscle fibers. The Journal of biophysical and biochemical cytology. 1961; 9: 493-5.
3. Chal J, Pourquie O. Making muscle: skeletal myogenesis in vivo and in vitro. Development. 2017; 144(12) :2104-22.  3. Shawl J, Pourquie O. Making muscle: skeletal myogenesis in vivo and in vitro. Development 2017; 144 (12): 2104-22.
4. Boldrin L, Muntoni F, Morgan JE. Are human and mouse satellite cells really the same? J Histochem Cytochem. 2010;58(11):941-55.  4. Boldrin L, Muntoni F, Morgan JE. Are human and mouse satellite cells really the same? J Histochem Cytochem. 2010; 58 (11): 941-55.
5. Negroni E, Gidaro T, Bigot A, Butler-Browne GS, Mouly V, Trollet C. Invited review: Stem cells and muscle diseases: advances in cell therapy strategies. Neuropathol Appl Neurobiol. 2015;41(3):270-87.  5. Negroni E, Gidaro T, Bigot A, Butler-Browne GS, Mouly V, Trollet C. Invited review: Stem cells and muscle diseases: advances in cell therapy strategies. Neuropathol Appl Neurobiol. 2015; 41 (3): 270-87.
6. Tucciarone L, Etxaniz U, Sandoná M, Consalvi S, Puri PL, Saccone V. Advanced Methods to Study the Cross Talk Between Fibro-Adipogenic Progenitors and Muscle Stem Cells. In: Bernardini C, editor. Duchenne Muscular Dystrophy: Methods and Protocols. New York, NY: Springer New York; 2018. p. 231-56.  6. Tucciarone L, Etxaniz U, Sandoná M, Consalvi S, Puri PL, Saccone V. Advanced Methods to Study the Cross Talk Between Fibro-Adipogenic Progenitors and Muscle Stem Cells. In: Bernardini C, editor. Duchenne Muscular Dystrophy: Methods and Protocols. New York, NY: Springer New York; 2018. p. 231-56.
7. Taniuchi M, Clark HB, Johnson EM. Induction of nerve growth factor receptor in Schwann cells after axotomy. Proceedings of the National Academy of Sciences of the United States of America. 1986;83(11):4094-8.  7. Taniuchi M, Clark HB, Johnson EM. Induction of nerve growth factor receptor in Schwann cells after axotomy. Proceedings of the National Academy of Sciences of the United States of America. 1986; 83 (11): 4094-8.
8. Tedesco FS, Moyle LA, Perdiguero E. Muscle Interstitial Cells: A Brief Field Guide to Non-satellite Cell Populations in Skeletal Muscle. In: Perdiguero E, Cornelison DDW, editors. Muscle Stem Cells: Methods and Protocols. New York, NY: Springer New York; 2017. p. 129-47.  8. Tedesco FS, Moyle LA, Perdiguero E. Muscle Interstitial Cells: A Brief Field Guide to Non-satellite Cell Populations in Skeletal Muscle. In: Perdiguero E, Cornelison DDW, editors. Muscle Stem Cells: Methods and Protocols. New York, NY: Springer New York; 2017. p. 129-47.
9. Zheng B, Cao B, Crisan M, Sun B, Li G, Logar A, et al. Prospective Identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol. 2007;25(9): 1025-34.  9. Zheng B, Cao B, Crisan M, Sun B, Li G, Logar A, et al. Prospective Identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol. 2007; 25 (9): 1025-34.
10. Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A, et al. Restoration of human dystrophin following transplantation of exon-skipping- engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell. 2007;1(6):646-57.  10. Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A, et al. Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell. 2007; 1 (6): 646-57.
11. Meng J, Chun S, Asfahani R, Lochmuller H, Muntoni F, Morgan J. Human skeletal muscle-derived CD133(+) cells form functional satellite cells after intramuscular transplantation in immunodeficient host mice. Mol Ther. 2014;22(5): 1008-17.  11. Meng J, Chun S, Asfahani R, Lochmuller H, Muntoni F, Morgan J. Human skeletal muscle-derived CD133 (+) cells form functional satellite cells after intramuscular transplantation in immunodeficient host mice. Mol Ther. 2014; 22 (5): 1008-17.
12. Negroni E, Riederer I, Chaouch S, Belicchi M, Razini P, Di Santo J, et al. In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study. Mol Ther. 2009;17(10):1771-8.  12. Negroni E, Riederer I, Chaouch S, Belicchi M, Razini P, Di Santo J, et al. In vivo myogenic potential of human CD133 + muscle-derived stem cells: a quantitative study. Mol Ther. 2009; 17 (10): 1771-8.
13. Torrente Y, Belicchi M, Marchesi C, D'Antona G, Cogiamanian F, Pisati F, et al. Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients. Cell Transplant. 2007;16(6):563-77.  13. Torrente Y, Belicchi M, Marchesi C, D'Antona G, Cogiamanian F, Pisati F, et al. Autologous transplantation of muscle-derived CD133 + stem cells in Duchenne muscle patients. Cell Transplant 2007; 16 (6): 563-77.
14. Vauchez K, Marolleau JP, Schmid M, Khattar P, Chapel A, Catelain C, et al. Aldehyde dehydrogenase activity identifies a population of human skeletal muscle cells with high myogenic capacities. Mol Ther. 2009;17(11):1948-58.  14. Vauchez K, Marolleau JP, Schmid M, Khattar P, Chapel A, Catelain C, et al. Aldehyde dehydrogenase activity identifies a population of human skeletal muscle cells with high myogenic capacities. Mol Ther. 2009; 17 (11): 1948-58.
15. Jean E, Laoudj-Chenivesse D, Notarnicola C, Rouger K, Serratrice N, Bonnieu A, et al. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells. J Cell Mol Med. 2011 ;15(1): 119-33. 16. Vella JB, Thompson SD, Bucsek MJ, Song M, Huard J. Murine and human myogenic cells identified by elevated aldehyde dehydrogenase activity: implications for muscle regeneration and repair. PLoS One. 2011 ;6(12):e29226. 15. Jean E, Laoudj-Chenivesse D, Notarnicola C, Rouger K, Serratrice N, Bonnieu A, et al. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells. J Cell Mol Med. 2011; 15 (1): 119-33. 16. Vella JB, Thompson SD, Bucsek MJ, Song M, Huard J. Murine and human myogenic cells identified by elevated aldehyde dehydrogenase activity: implications for muscle regeneration and repair. PLoS One. 2011; 6 (12): e29226.
17. Pisani DF, Clement N, Loubat A, Plaisant M, Sacconi S, Kurzenne JY, et al. Hierarchization of myogenic and adipogenic progenitors within human skeletal muscle. Stem Cells. 2010;28(12):2182-94.  17. Pisani DF, Clement N, Loubat A, Plaisant M, Sacconi S, Kurzenne JY, et al. Hierarchization of myogenic and adipogenic progenitors within human skeletal muscle. Stem Cells 2010; 28 (12): 2182-94.
18. Pisani DF, Dechesne CA, Sacconi S, Delplace S, Belmonte N, Cochet O, et al. Isolation of a highly myogenic CD34-negative subset of human skeletal muscle cells free of adipogenic potential. Stem Cells. 2010;28(4):753-64.  18. Pisani DF, Dechesne CA, Sacconi S, Delplace S, Belmonte N, Cochet O, et al. Isolation of a highly myogenic CD34-negative subset of human skeletal muscle cells free of adipogenic potential. Stem Cells 2010; 28 (4): 753-64.
19. Lecourt S, Marolleau JP, Fromigue O, Vauchez K, Andriamanalijaona R, Ternaux B, et al. Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro. Exp Cell Res. 2010;316(15):2513-26.  19. Lecourt S, Marolleau JP, Fromigue O, Vauchez K, Andriamanalijaona R, Ternaux B, et al. Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro. Exp Cell Res. 2010; 316 (15): 2513-26.
20. Castiglioni A, Hettmer S, Lynes MD, Rao TN, Tchessalova D, Sinha I, et al. Isolation of Progenitors that Exhibit Myogenic/Osteogenic Bipotency In Vitro by Fluorescence-Activated Cell Sorting from Human Fetal Muscle. Stem Cell Reports. 2014;2(1):92-106.  20. Castiglioni A, Hettmer S, Lynes MD, Rao TN, Tchessalova D, Sinha I, et al. Isolation of Progenitors that Exhibit Myogenic / Osteogenic Bipotency In Vitro by Fluorescence-Activated Cell Sorting from Human Fetal Muscle. Stem Cell Reports. 2014; 2 (1): 92-106.
21. Bareja A, Holt JA, Luo G, Chang C, Lin J, Hinken AC, et al. Human and mouse skeletal muscle stem cells: convergent and divergent mechanisms of myogenesis. PLoS One. 2014;9(2):e90398.  21. Bareja A, Holt JA, Luo G, Chang C, Lin J, Hinken AC, et al. Human and mouse skeletal muscle stem cells: convergent and divergent mechanisms of myogenesis. PLoS One. 2014; 9 (2): e90398.
22. Tamaki T, Uchiyama Y, Hirata M, Hashimoto H, Nakajima N, Saito K, et al. Therapeutic isolation and expansión of human skeletal muscle-derived stem cells for the use of muscle-nerve-blood vessel reconstitution. Front Physiol. 2015;6:165.  22. Tamaki T, Uchiyama Y, Hirata M, Hashimoto H, Nakajima N, Saito K, et al. Therapeutic isolation and expansion of human skeletal muscle-derived stem cells for the use of muscle-nerve-blood vessel reconstitution. Front Physiol 2015; 6: 165.
23. Xu X, Wilschut KJ, Kouklis G, Tian H, Hesse R, Garland C, et al. Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles. Stem Cell Reports. 2015;5(3):419-34.  23. Xu X, Wilschut KJ, Kouklis G, Tian H, Hesse R, Garland C, et al. Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles. Stem Cell Reports. 2015; 5 (3): 419-34.
24. Charville GW, Cheung TH, Yoo B, Santos PJ, Lee GK, Shrager JB, et al. Ex Vivo Expansión and In Vivo Self-Renewal of Human Muscle Stem Cells. Stem Cell Reports. 2015;5(4):621-32.  24. Charville GW, Cheung TH, Yoo B, Santos PJ, Lee GK, Shrager JB, et al. Ex Vivo Expansión and In Vivo Self-Renewal of Human Muscle Stem Cells. Stem Cell Reports. 2015; 5 (4): 621-32.
25. Alexander MS, Rozkalne A, Colletta A, Spinazzola JM, Johnson S, Rahimov F, et al. CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies. Cell Stem Cell. 2016;19(6):800-7.  25. Alexander MS, Rozkalne A, Colletta A, Spinazzola JM, Johnson S, Rahimov F, et al. CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies. Cell Stem Cell. 2016; 19 (6): 800-7.
26. Uezumi A, Nakatani M, Ikemoto-Uezumi M, Yamamoto N, Morita M, Yamaguchi A, et al. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle. Stem Cell Reports. 2016;7(2):263-78.  26. Uezumi A, Nakatani M, Ikemoto-Uezumi M, Yamamoto N, Morita M, Yamaguchi A, et al. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle. Stem Cell Reports. 2016; 7 (2): 263-78.
27. Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science. 2010;329(5995):1078-81.  27. Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 2010; 329 (5995): 1078-81.
28. Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A, et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science. 2005;309(5743):2064-7.  28. Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A, et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005; 309 (5743): 2064-7.
29. Brimah K, Ehrhardt J, Mouly V, Butler-Browne GS, Partridge TA, Morgan JE. Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors. Hum Gene Ther. 2004; 15(11): 1109-24.  29. Brimah K, Ehrhardt J, Mouly V, Butler-Browne GS, Partridge TA, Morgan JE. Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors. Hum Gene Ther. 2004; 15 (11): 1109-24.
30. Cooper RN, Irintchev A, Di Santo JP, Zweyer M, Morgan JE, Partridge TA, et al. A new immunodeficient mouse model for human myoblast transplantation. Hum Gene Ther. 2001 ;12(7):823-31.  30. Cooper RN, Irintchev A, Di Santo JP, Zweyer M, Morgan JE, Partridge TA, et al. A new immunodeficient mouse model for human myoblast transplantation. Hum Gene Ther. 2001; 12 (7): 823-31.
31. Bareja A, Billin AN. Satellite cell therapy - from mice to men. Skeletal muscle. 2013;3(1):2. 32. Partridge TA, Grounds M, Sloper JC. Evidence of fusión between host and donor myoblasts in skeletal muscle grafts. Nature. 1978;273(5660):306-8. 31. Bareja A, Billin AN. Satellite cell therapy - from mice to men. Skeletal muscle. 2013; 3 (1): 2. 32. Partridge TA, Grounds M, Sloper JC. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature 1978; 273 (5660): 306-8.
33. Stadler G, Chen JC, Wagner K, Robín JD, Shay JW, Emerson Jr. CP, et al. Establish ent of clonal yogenic cell lines fro severely affected dystrophic muscles - CDK4 aintains the myogenic population. Skeletal muscle. 2011 ; 1 (1 ): 12.  33. Stadler G, Chen JC, Wagner K, Robin JD, Shay JW, Emerson Jr. CP, et al. Establish ent of clonal yogenic cell lines fro severely affected dystrophic muscles - CDK4 aintains the myogenic population. Skeletal muscle. 2011; 1 (1): 12.
34. Konigsberg IR. The differentiation of cross-striated myofibrils in short term cell culture. Exp Cell Res. 1960;21 :414-20.  34. Konigsberg IR. The differentiation of cross-striated myofibrils in short term cell culture. Exp Cell Res. 1960; 21: 414-20.
35. Scott IC, Tomlinson W, Walding A, Isherwood B, Dougall IG. Large-scale isolation of human skeletal muscle satellite cells from post-mortem tissue and development of quantitative assays to evalúate modulators of myogenesis. J Cachexia Sarcopenia Muscle. 2013;4(2): 157-69.  35. Scott IC, Tomlinson W, Walding A, Isherwood B, Dougall IG. Large-scale isolation of human skeletal muscle satellite cells from post-mortem tissue and development of quantitative assays to evaluate modulators of myogenesis. J Cachexia Sarcopenia Muscle. 2013; 4 (2): 157-69.
36. Briggs D, Morgan JE. Recent progress in satellite cell/myoblast engraftment -- relevance for therapy. FEBS J. 2013;280(17):4281-93.  36. Briggs D, Morgan JE. Recent progress in satellite cell / myoblast engraftment - relevance for therapy. FEBS J. 2013; 280 (17): 4281-93.
37. Cossu G, Sampaolesi M. New therapies for Duchenne muscular dystrophy: challenges, prospects and clinical triáis. Trends Mol Med. 2007;13(12):520-6.  37. Cossu G, Sampaolesi M. New therapies for Duchenne muscular dystrophy: challenges, prospects and clinical triáis. Trends Mol Med. 2007; 13 (12): 520-6.
38. Mouly V, Aamiri A, Perie S, Mamchaoui K, Barani A, Bigot A, et al. Myoblast transfer therapy: is there any light at the end of the tunnel? Acta Myol. 2005;24(2):128- 33.  38. Mouly V, Aamiri A, Perie S, Mamchaoui K, Barani A, Bigot A, et al. Myoblast transfer therapy: is there any light at the end of the tunnel? Myol Act. 2005; 24 (2): 128-33.
39. Negroni E, Butler-Browne GS, Mouly V. Myogenic stem cells: regeneration and cell therapy in human skeletal muscle. Pathol Biol (París). 2006;54(2): 100-8.  39. Negroni E, Butler-Browne GS, Mouly V. Myogenic stem cells: regeneration and cell therapy in human skeletal muscle. Pathol Biol (Paris). 2006; 54 (2): 100-8.
40. 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.  40. 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.
41. Cooper RN, Thiesson D, Furling D, Di Santo JP, Butler-Browne GS, Mouly V. Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation. Hum Gene Ther. 2003;14(12):1169-79. 41. Cooper RN, Thiesson D, Furling D, Di Santo JP, Butler-Browne GS, Mouly V. Extended amplification in vitro and replicative senescence: key factors implied in the success of human myoblast transplantation. Hum Gene Ther. 2003; 14 (12): 1169-79.
42. Motohashi N, Asakura A. Muscle satellite cell heterogeneity and self-renewal. Front Cell Dev Biol. 2014;2:1. 42. Motohashi N, Asakura A. Muscle satellite cell heterogeneity and self-renewal. Front Cell Dev Biol. 2014; 2: 1.
43. Shadrin IY, Khodabukus A, Bursac N. Striated muscle function, regeneration, and repair. Cell Mol Ufe Sci. 2016;73(22):4175-202.  43. Shadrin IY, Khodabukus A, Bursac N. Striated muscle function, regeneration, and repair. Cell Mol Ufe Sci. 2016; 73 (22): 4175-202.
44. Urbani L, Piccoli M, Franzin C, Pozzobon M, De Coppi P. Hypoxia increases mouse satellite cell clone proliferation maintaining both in vitro and in vivo heterogeneity and myogenic potential. PLoS One. 2012;7(11):e49860.  44. Urbani L, Piccoli M, Franzin C, Pozzobon M, De Coppi P. Hypoxia increases mouse satellite cell clone proliferation maintaining both in vitro and in vivo heterogeneity and myogenic potential. PLoS One. 2012; 7 (11): e49860.
45. Miersch C, Stange K, Hering S, Kolisek M, Viergutz T, Rontgen M. Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile. Sci Rep. 2017;7:45052.  45. Miersch C, Stange K, Hering S, Kolisek M, Viergutz T, Rontgen M. Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile. Sci Rep. 2017; 7: 45052.
46. Shan T, Xu Z, Liu J, Wu W, Wang Y. Lkb1 regulation of skeletal muscle development, metabolism and muscle progenitor cell homeostasis. J Cell Physiol. 2017;232(10):2653-6.  46. Shan T, Xu Z, Liu J, Wu W, Wang Y. Lkb1 regulation of skeletal muscle development, metabolism and muscle progenitor cell homeostasis. J Cell Physiol. 2017; 232 (10): 2653-6.
47. Theret M, Gsaier L, Schaffer B, Juban G, Ben Larbi S, Weiss-Gayet M, et al. AMPKalpha1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis. EMBO J. 2017;36(13):1946-62.  47. Theret M, Gsaier L, Schaffer B, Juban G, Ben Larbi S, Weiss-Gayet M, et al. AMPKalpha1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis. EMBO J. 2017; 36 (13): 1946-62.
48. Alonso-Martin S, Rochat A, Mademtzoglou D, Moráis J, de Reynies A, Aurade F, et al. Gene Expression Profiling of Muscle Stem Cells Identifies Novel Regulators of Postnatal Myogenesis. Front Cell Dev Biol. 2016;4:58.  48. Alonso-Martin S, Rochat A, Mademtzoglou D, Moráis J, of Reynies A, Aurade F, et al. Gene Expression Profiling of Muscle Stem Cells Identifies Novel Regulators of Postnatal Myogenesis. Front Cell Dev Biol. 2016; 4: 58.
49. Pawlikowski B, Vogler TO, Gadek K, Olwin BB. Regulation of skeletal muscle stem cells by fibroblast growth factors. Dev Dyn. 2017;246(5):359-67. 50. Shan T, Xu Z, Wu W, Liu J, Wang Y. Roles of Notchl Signaling in Regulating Satellite Cell Fates Choices and Postnatal Skeletal Myogenesis. J Cell Physiol. 2017;232(11):2964-7. 49. Pawlikowski B, Vogler TO, Gadek K, Olwin BB. Regulation of skeletal muscle stem cells by fibroblast growth factors. Dev Dyn 2017; 246 (5): 359-67. 50. Shan T, Xu Z, Wu W, Liu J, Wang Y. Roles of Notchl Signaling in Regulating Satellite Cell Fates Choices and Postnatal Skeletal Myogenesis. J Cell Physiol. 2017; 232 (11): 2964-7.
51. Rajgara RF, Lala-Tabbert N, Marchildon F, Lamarche E, MacDonald JK, Scott DA, et al. SOX7 Is Required for Muscle Satellite Cell Development and Maintenance. Stem Cell Reports. 2017.  51. Rajgara RF, Lala-Tabbert N, Marchildon F, Lamarche E, MacDonald JK, Scott DA, et al. SOX7 Is Required for Muscle Satellite Cell Development and Maintenance. Stem Cell Reports. 2017
52. Sun C, De Mello V, Mohamed A, Ortuste Quiroga HP, Garcia-Munoz A, Al Bloshi A, et al. Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function. Stem Cells. 2017; 35(8): 1958-72.  52. Sun C, De Mello V, Mohamed A, Ortuste Quiroga HP, Garcia-Munoz A, Al Bloshi A, et al. Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function. Stem Cells 2017; 35 (8): 1958-72.
53. París ND, Soroka A, Klose A, Liu W, Chakkalakal JV. Smad4 restricts differentiation to promote expansión of satellite cell derived progenitors during skeletal muscle regeneration. Elife. 2016;5.  53. Paris ND, Soroka A, Klose A, Liu W, Chakkalakal JV. Smad4 restrictedts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration. Elife 2016; 5.
54. Borisov AB. Regeneration of skeletal and cardiac muscle in mammals: do nonprimate models resemble human pathology? Wound Repair Regen. 1999;7(1):26- 35.  54. Borisov AB. Regeneration of skeletal and cardiac muscle in mammals: do nonprimate models resemble human pathology? Wound Repair Regen. 1999; 7 (1): 26-35.
55. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol. 1999; 144(6): 1113-22.  55. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol. 1999; 144 (6): 1113-22.
56. Ono Y, Masuda S, Nam HS, Benezra R, Miyagoe-Suzuki Y, Takeda S. Slow- dividing satellite cells retain long-term self-renewal ability in adult muscle. J Cell Sci. 2012;125(Pt 5):1309-17.  56. Ono Y, Masuda S, Nam HS, Benezra R, Miyagoe-Suzuki Y, Takeda S. Slow- dividing satellite cells retain long-term self-renewal ability in adult muscle. J Cell Sci. 2012; 125 (Pt 5): 1309-17.
57. Huard J, Verreault S, Roy R, Tremblay M, Tremblay JP. High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice. The Journal of clinical investigation. 1994;93(2):586-99.  57. Huard J, Verreault S, Roy R, Tremblay M, Tremblay JP. High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice. The Journal of clinical investigation. 1994; 93 (2): 586-99.
58. Skuk D, Paradis M, Goulet M, Chapdelaine P, Rothstein DM, Tremblay JP. Intramuscular transplantation of human postnatal myoblasts generates functional donor-derived satellite cells. Mol Ther. 2010;18(9):1689-97.  58. Skuk D, Paradis M, Goulet M, Chapdelaine P, Rothstein DM, Tremblay JP. Intramuscular transplantation of human postnatal myoblasts generates functional donor-derived satellite cells. Mol Ther. 2010; 18 (9): 1689-97.
59. Laumonier T, Bermont F, Hoffmeyer P, Kindler V, Menetrey J. Human myogenic reserve cells are quiescent stem cells that contribute to muscle regeneration after intramuscular transplantation in immunodeficient mice. Sci Rep. 2017;7(1):3462.  59. Laumonier T, Bermont F, Hoffmeyer P, Kindler V, Menetrey J. Human myogenic reserve cells are quiescent stem cells that contribute to muscle regeneration after intramuscular transplantation in immunodeficient mice. Sci Rep. 2017; 7 (1): 3462.
60. Perie S, Trollet C, Mouly V, Vanneaux V, Mamchaoui K, Bouazza B, et al. Autologous myoblast transplantation for oculopharyngeal muscular dystrophy: a phase l/lla clinical study. Mol Ther. 2014;22(1):219-25.  60. Perie S, Trollet C, Mouly V, Vanneaux V, Mamchaoui K, Bouazza B, et al. Autologous myoblast transplantation for oculopharyngeal muscular dystrophy: a phase l / lla clinical study. Mol Ther. 2014; 22 (1): 219-25.
61. El Haddad M, Notarnicola C, Evano B, El Khatib N, Blaquiere M, Bonnieu A, et al. Retinóle acid maintains human skeletal muscle progenitor cells in an immature State. Cell Mol Ufe Sci. 2017;74(10): 1923-36.  61. Haddad M, Notarnicola C, Evano B, El Khatib N, Blaquiere M, Bonnieu A, et al. Retinóle acid maintains human skeletal muscle progenitor cells in an immature State. Cell Mol Ufe Sci. 2017; 74 (10): 1923-36.
62. Nierobisz LS, Cheatham B, Buehrer BM, Sexton JZ. High-content screening of human primary muscle satellite cells for new therapies for muscular atrophy/dystrophy. Curr Chem Genom Transí Med. 2013;7:21-9.  62. Nierobisz LS, Cheatham B, Buehrer BM, Sexton JZ. High-content screening of human primary muscle satellite cells for new therapies for muscular atrophy / dystrophy. Curr Chem Genom Transí Med. 2013; 7: 21-9.
63. Sarig R, Baruchi Z, Fuchs O, Nudel U, Yaffe D. Regeneration and transdifferentiation potential of muscle-derived stem cells propagated as myospheres. Stem Cells. 2006;24(7): 1769-78.  63. Sarig R, Baruchi Z, Fuchs O, Nudel U, Yaffe D. Regeneration and transdifferentiation potential of muscle-derived stem cells propagated as myospheres. Stem Cells 2006; 24 (7): 1769-78.
64. Westerman KA. Myospheres are composed of two cell types: one that is myogenic and a second that is mesenchymal. PLoS One. 2015;10(2):e0116956.  64. Westerman KA. Myospheres are composed of two cell types: one that is myogenic and a second that is mesenchymal. PLoS One. 2015; 10 (2): e0116956.
65. Westerman KA, Penvose A, Yang Z, Alien PD, Vacanti CA. Adult muscle 'stem' cells can be sustained in culture as free-floating myospheres. Exp Cell Res. 2010;316(12): 1966-76. 66. Naldaiz-Gastesi N, Goicoechea M, Alonso-Martin S, Aiastui A, Lopez-Mayorga M, Garcia-Belda P, et al. Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells. Stem Cell Reports. 2016;7(3):411-24. 65. Westerman KA, Penvose A, Yang Z, Alien PD, Vacanti CA. Adult muscle 'stem' cells can be sustained in culture as free-floating myospheres. Exp Cell Res. 2010; 316 (12): 1966-76. 66. Naldaiz-Gastesi N, Goicoechea M, Alonso-Martin S, Aiastui A, Lopez-Mayorga M, Garcia-Belda P, et al. Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells. Stem Cell Reports. 2016; 7 (3): 411-24.
67. Nomura T, Ashihara E, Tateishi K, Ueyama T, Takahas-Hi T, Yamagishi M, et al. Therapeutic potential of stem/progenitor cells in human skeletal muscle for cardiovascular regeneration. Curr Stem Cell Res Ther. 2007;2(4):293-300.  67. Nomura T, Ashihara E, Tateishi K, Ueyama T, Takahas-Hi T, Yamagishi M, et al. Therapeutic potential of stem / progenitor cells in human skeletal muscle for cardiovascular regeneration. Curr Stem Cell Res Ther. 2007; 2 (4): 293-300.
68. Wei Y, Li Y, Chen C, Stoelzel K, Kauf ann AM, Albers AE. Human skeletal muscle-derived stem cells retain stem cell properties after expansión in myosphere culture. Exp Cell Res. 2011 ;317(7): 1016-27.  68. Wei Y, Li Y, Chen C, Stoelzel K, Kauf ann AM, Albers AE. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture. Exp Cell Res. 2011; 317 (7): 1016-27.
69. Jungbluth H, Ochala J, Treves S, Gautel M. Current and future therapeutic approaches to the congenital myopathies. Semin Cell Dev Biol. 2017;64:191-200.  69. Jungbluth H, Ochala J, Treves S, Gautel M. Current and future therapeutic approaches to the congenital myopathies. Semin Cell Dev Biol. 2017; 64: 191-200.
70. Emery AE. The muscular dystrophies. Lancet. 2002;359(9307):687-95.  70. Emery AE. The muscular dystrophies. Lancet 2002; 359 (9307): 687-95.
71. Telukuntla KS, Suncion VY, Schulman IH, Haré JM. The advancing field of cell- based therapy: insights and lessons from clinical triáis. J Am Heart Assoc. 2013;2(5):e000338.  71. Telukuntla KS, Suncion VY, Schulman IH, Haé JM. The advancing field of cell- based therapy: insights and lessons from clinical triáis. J Am Heart Assoc. 2013; 2 (5): e000338.
72. Snijders T, Parise G. Role of muscle stem cells in sarcopenia. Curr Opin Clin Nutr Metab Care. 2017;20(3): 186-90.  72. Snijders T, Parise G. Role of muscle stem cells in sarcopenia. Curr Opin Clin Nutr Metab Care. 2017; 20 (3): 186-90.
73. Williams JK, Dean A, Badlani G, Andersson KE. Regenerative Medicine Therapies for Stress Urinary Incontinence. J Urol. 2016;196(6):1619-26.  73. Williams JK, Dean A, Badlani G, Andersson KE. Regenerative Medicine Therapies for Stress Urinary Incontinence. J Urol. 2016; 196 (6): 1619-26.
74. Gras S, Tolstrup CK, Lose G. Regenerative medicine provides alternative strategies for the treatment of anal incontinence. Int Urogynecol J. 2017;28(3):341-50. 74. Gras S, Tolstrup CK, Lose G. Regenerative medicine provides alternative strategies for the treatment of anal incontinence. Int Urogynecol J. 2017; 28 (3): 341-50.
75. Hart ML, Izeta A, Herrera-I mbroda B, Amend B, Brinchmann JE. Cell Therapy for Stress Urinary Incontinence. Tissue Eng Part B Rev. 2015;21(4):365-76. 75. Hart ML, Izeta A, Herrera-I mbroda B, Amend B, Brinchmann JE. Cell Therapy for Stress Urinary Incontinence. Tissue Eng Part B Rev. 2015; 21 (4): 365-76.
76. Herrera-I mbroda B, Lara MF, Izeta A, Sievert KD, Hart ML. Stress urinary incontinence animal models as a tool to study cell-based regenerative therapies targeting the urethral sphincter. Adv Drug Deliv Rev. 2015;82-83:106-16.  76. Herrera-I mbroda B, Lara MF, Izeta A, Sievert KD, Hart ML. Stress urinary incontinence animal models as a tool to study cell-based regenerative therapies targeting the urethral sphincter. Adv Drug Deliv Rev. 2015; 82-83: 106-16.
77. Kivela R, Salmela I, Nguyen YH, Petrova TV, Koistinen HA, Wiener Z, et al. The transcription factor Proxl is essential for satellite cell differentiation and muscle fibre- type regulation. Nat Commun. 2016;7:13124.  77. Kivela R, Salmela I, Nguyen YH, Petrova TV, Koistinen HA, Wiener Z, et al. The transcription factor Proxl is essential for satellite cell differentiation and muscle fiber type regulation. Nat Commun 2016; 7: 13124.
78. Sumino Y, Hirata Y, Hanada M, Akita Y, Sato F, Mimata H. Long-term cryopreservation of pyramidalis muscle specimens as a source of striated muscle stem cells for treatment of post-prostatectomy stress urinary incontinence. Prostate. 2011 ;71 (11): 1225-30.  78. Sumino Y, Hirata Y, Hanada M, Akita Y, Sato F, Mimata H. Long-term cryopreservation of pyramidalis muscle specimens as a source of striated muscle stem cells for treatment of post-prostatectomy stress urinary incontinence. Prostate 2011; 71 (11): 1225-30.
79. Sharifiaghdas F, Taheri M, Moghadasali R. Isolation of human adult stem cells from muscle biopsy for future treatment of urinary incontinence. Urol J. 2011 ;8(1 ):54-9. 79. Sharifiaghdas F, Taheri M, Moghadasali R. Isolation of human adult stem cells from muscle biopsy for future treatment of urinary incontinence. Urol J. 2011; 8 (1): 54-9.
80. Vinarov A, Atala A, Yoo J, Slusarenco R, Zhumtaev M, Zhito A, et al. Cell therapy for stress urinary incontinence: Present-day frontiers. J Tissue Eng Regen Med. 2017. 80. Vinarov A, Atala A, Yoo J, Slusarenco R, Zhumtaev M, Zhito A, et al. Cell therapy for stress urinary incontinence: Present-day frontiers. J Tissue Eng Regen Med. 2017.
81. Kayalioglu G, Altay B, Uyaroglu FG, Bademkiran F, Uludag B, Ertekin C. Morphology and innervation of the human cremaster muscle in relation to its function. Anat Rec (Hoboken). 2008;291(7):790-6.  81. Kayalioglu G, Altay B, Uyaroglu FG, Bademkiran F, Uludag B, Ertekin C. Morphology and innervation of the human cremaster muscle in relation to its function. Anat Rec (Hoboken). 2008; 291 (7): 790-6.
82. 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.  82. 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.
83. Harnaen EJ, Na AF, Shenker NS, Sourial M, Farmer PJ, Southwell BR, et al. The anatomy of the cremaster muscle during inguinoscrotal testicular descent in the rat. J Pediatr Surg. 2007;42(12):1982-7. 84. Lie G, Hutson JM. The role of cremaster muscle in testicular descent in humans and animal odels. Pediatr Surg Int. 2011 ;27(12): 1255-65. 83. Harnaen EJ, Na AF, Shenker NS, Sourial M, Farmer PJ, Southwell BR, et al. The anatomy of the cremaster muscle during inguinoscrotal testicular descent in the rat. J Pediatr Surg. 2007; 42 (12): 1982-7. 84. Lie G, Hutson JM. The role of cremaster muscle in testicular descent in humans and animal odels. Pediatr Surg Int. 2011; 27 (12): 1255-65.
85. Sanders N, Buraundi S, Balic A, Southwell BR, Hutson JM. Cremaster muscle myogenesis in the tip of the rat gubernaculum supports active gubernacular elongation during inguinoscrotal testicular descent. J Urol. 2011 ;186(4 Suppl):1606-13.  85. Sanders N, Buraundi S, Balic A, Southwell BR, Hutson JM. Cremaster muscle myogenesis in the tip of the rat goverculum supports active governmentalcular elongation during inguinoscrotal testicular descent. J Urol. 2011; 186 (4 Suppl): 1606-13.
86. Tanyel FC, Talim B, Atilla P, Muftuoglu S, Kale G. Myogenesis within the human gubernaculum: histological and immunohistochemical evaluation. Eur J Pediatr Surg. 2005; 15(3): 175-9.  86. Tanyel FC, Talim B, Atilla P, Muftuoglu S, Kale G. Myogenesis within the human governmentalculum: histological and immunohistochemical evaluation. Eur J Pediatr Surg. 2005; 15 (3): 175-9.
87. Borirakchanyavat S, Baskin LS, Kogan BA, Cunha GR. Smooth and striated muscle development in the intrinsic urethral sphincter. J Urol. 1997; 158(3 Pt 2): 1119- 87. Borirakchanyavat S, Baskin LS, Kogan BA, Cunha GR. Smooth and striated muscle development in the intrinsic urethral sphincter. J Urol. 1997; 158 (3 Pt 2): 1119-
22. 22
88. García-Parra P, Naldaiz-Gastesi N, Maroto M, Padin JF, Goicoechea M, Aiastui A, et al. Murine muscle engineered from dermal precursors: an in vitro model for skeletal muscle generation, degeneration, and fatty infiltration. Tissue engineering. 2014;20(1):28-41.  88. García-Parra P, Naldaiz-Gastesi N, Maroto M, Padin JF, Goicoechea M, Aiastui A, et al. Murine muscle engineered from dermal precursors: an in vitro model for skeletal muscle generation, degeneration, and fatty infiltration. Tissue engineering 2014; 20 (1): 28-41.

Claims

REIVINDICACIONES
1. Composición que comprende una población celular de células precursoras multipotentes aisladas del músculo cremáster humano, donde dicha población celular comprende al menos un 20% de células precursoras multipotentes aisladas del músculo cremáster humano. 1. Composition comprising a cell population of multipotent precursor cells isolated from the human cremaster muscle, wherein said cell population comprises at least 20% of multipotent precursor cells isolated from the human cremaster muscle.
2. Composición según la reivindicación 1 , donde dicha población celular es positiva para los marcadores: Pax7, MyoD y MyHC. 2. Composition according to claim 1, wherein said cell population is positive for the markers: Pax7, MyoD and MyHC.
3. Composición que comprende mioesferas obtenidas u obtenibles de cultivos de células precursoras multipotentes aisladas del músculo cremáster humano, donde dichas miosferas se caracterizan por comprender células precursoras miogénicas positivas para los marcadores: Pax7, MyoD y MyHC. 3. Composition comprising myospheres obtained or obtainable from cultures of multipotent precursor cells isolated from human cremater muscle, where said myospheres are characterized by comprising myogenic precursor cells positive for the markers: Pax7, MyoD and MyHC.
4. Composición que comprende una población celular de células miogénicas obtenidas a partir de la diferenciación de células precursoras multipotentes aisladas del músculo cremáster humano, caracterizada porque dicha población comprende al menos un 20% de células miogénicas caracterizadas porque expresan los genes miogénicos Pax7, Myf5, MyoD1 , Myogenin, MyHC3 y MyHC2, en mayor cantidad comparada con un control negativo. 4. Composition comprising a cellular population of myogenic cells obtained from the differentiation of multipotent precursor cells isolated from human cremaster muscle, characterized in that said population comprises at least 20% of myogenic cells characterized in that they express the myogenic genes Pax7, Myf5, MyoD1, Myogenin, MyHC3 and MyHC2, in greater quantity compared to a negative control.
5. Composición que comprende una población celular de células precursoras vasculogénicas obtenidas o aisladas a partir de células precursoras multipotentes aisladas del músculo cremáster humano, caracterizada porque dicha población comprende al menos un 20% de células precursoras vasculogénicas caracterizadas porque son positivas para los marcadores: p75NTR, SOX2, PAX3, DLK1 , RGS5, AOC3, ITGA7, ECRG4 y negativas para el marcador CD56. 5. Composition comprising a cell population of vasculogenic precursor cells obtained or isolated from multipotent precursor cells isolated from human cremaster muscle, characterized in that said population comprises at least 20% of vasculogenic precursor cells characterized in that they are positive for markers: p75NTR , SOX2, PAX3, DLK1, RGS5, AOC3, ITGA7, ECRG4 and negative for the CD56 marker.
6. Composición que comprende una población celular de células precursoras neurogénicas obtenidas o aisladas a partir de células precursoras multipotentes aisladas del músculo cremáster humano, caracterizada porque dicha población comprende al menos un 20% de células precursoras neurogénicas caracterizadas porque son positivas para los marcadores: p75NTR, SOX2, PAX3, CD56, SOX10, ERBB3, L1CAM, CDH2, CDH19, PMP22, PLP1 , DHH y negativas para el marcador SOX9. 6. Composition comprising a cell population of neurogenic precursor cells obtained or isolated from multipotent precursor cells isolated from human crema muscle, characterized in that said population comprises at least 20% of neurogenic precursor cells characterized in that they are positive for markers: p75NTR , SOX2, PAX3, CD56, SOX10, ERBB3, L1CAM, CDH2, CDH19, PMP22, PLP1, DHH and negative for the SOX9 marker.
7. La composición según cualquiera de las reivindicaciones anteriores, o cualquiera de sus combinaciones, donde dicha composición es una composición farmacéutica. 7. The composition according to any of the preceding claims, or any combination thereof, wherein said composition is a pharmaceutical composition.
8.- La composición farmacéutica según la reivindicación 7, donde dicha composición además comprende un vehículo farmacéuticamente aceptable. 8. The pharmaceutical composition according to claim 7, wherein said composition further comprises a pharmaceutically acceptable carrier.
9. La composición farmacéutica según la reivindicación 7 u 8, donde dicha composición además comprende otro principio activo. 9. The pharmaceutical composition according to claim 7 or 8, wherein said composition further comprises another active ingredient.
10. La composición farmacéutica según cualquiera de las reivindicaciones 7 a 9, donde dicha composición farmacéutica se prepara en una forma de dosificación líquida, sólida o semisólida, preferiblemente en forma de suspensión, para su uso en la administración mediante la implantación, inyección o infusión en un sujeto que necesita tratamiento. 10. The pharmaceutical composition according to any of claims 7 to 9, wherein said pharmaceutical composition is prepared in a liquid, solid or semi-solid dosage form, preferably in suspension form, for use in administration by implantation, injection or infusion in a subject that needs treatment.
11. La composición farmacéutica según cualquiera de las reivindicaciones 7 a 9, donde dicha composición farmacéutica se prepara en una forma de suspensión estéril con un excipiente farmacéuticamente aceptable, para su uso en la administración a un sujeto por vía parenteral. 11. The pharmaceutical composition according to any of claims 7 to 9, wherein said pharmaceutical composition is prepared in a sterile suspension form with a pharmaceutically acceptable excipient, for use in administration to a subject parenterally.
12. Dispositivo médico que comprende la composición según cualquiera de las reivindicaciones 1 a 11. 12. Medical device comprising the composition according to any of claims 1 to 11.
13. La composición según se describe en cualquiera de las reivindicaciones 1 a 7, o el dispositivo médico según la reivindicación 12, para su uso en medicina. 13. The composition as described in any one of claims 1 to 7, or the medical device according to claim 12, for use in medicine.
14. La composición según se describe en cualquiera de las reivindicaciones 1 a 11 , o el dispositivo médico según la reivindicación 12, para su uso en aumentar parcial o completamente, restaurar o reemplazar la actividad funcional de un tejido u órgano enfermo o dañado. 14. The composition as described in any one of claims 1 to 11, or the medical device according to claim 12, for use in partially or completely increasing, restoring or replacing the functional activity of a diseased or damaged tissue or organ.
15. La composición según se describe en cualquiera de las reivindicaciones 1 a 11 , o el dispositivo médico según la reivindicación 11 , para su uso en el tratamiento de la incontinencia urinaria, la incontinencia fecal, o ambas. 15. The composition as described in any one of claims 1 to 11, or the medical device according to claim 11, for use in the treatment of urinary incontinence, fecal incontinence, or both.
16. La composición según se describe en cualquiera de las reivindicaciones 1 a 11 , o el dispositivo médico según la reivindicación 11 , para su uso en la regeneración del músculo estriado. 16. The composition as described in any one of claims 1 to 11, or the medical device according to claim 11, for use in the regeneration of striated muscle.
PCT/ES2019/070167 2018-03-12 2019-03-12 Multipotent precursor cells obtained from the cremaster muscle and use thereof to develop therapies and regenerative medicine WO2019175459A1 (en)

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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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