Methods for obtaining myogenic cells

Abstract

The present invention relates to a method for obtaining skeletal muscle derived cells (SMDC), and to the use of SMDC in a method for preventing and / or treating neuromyopathy and / or myopathy, wherein the neuromyopathy and / or myopathy is incontinence, in particular urinary incontinence and / or anal incontinence or fecal incontinence.

Description

[0001] The present invention relates to a method for obtaining skeletal muscle derived cells (SMDC), and the use of SMDC in a method for improving neuromuscular connection and for treating muscle dysfunction, wherein the muscle dysfunction is incontinence, in particular urinary and / or anal incontinence.

[0002] The ability to maintain continence is essential for our well-being as social beings. Loss of anal continence leads to physical, physiological and social impairments. It is often assumed that mainly elderly and disabled people suffer from anal incontinence, however, these symptoms may occur in people of all ages. Anal incontinence (i.e. loss of control over the contents of the intestine) ranges from small traces of stool in underwear to flatulence to large uncontrolled passages of soft or solid stools. Its causes can be multi-layered and complex. In addition to the extremely impaired quality of life of the affected individuals, the cost factor to the public health system caused by loss of anal continence cannot be underestimated. In addition, anal incontinence is the second most common reason for hospitalization in nursing homes (more common than dementia). One in three elderly people in nursing homes or hospitals suffer from fecal incontinence.

[0003] Two skeletal muscles are important for voluntary control of the contraceptive organs: the external anal sphincter (Musculus sphincterani externus) and the puborectalis, which is part of the levator ani (Musculus sphincter ani externus). The remaining pelvic diaphragm (M. pubococcygeus, M. pubococcygeus, M. iliococcygeus) may play a more or less supporting role. The external anal sphincter is innervated by the pudendal nerve (N. pudendus). Both the external anal sphincter and the puborectalis can maintain a constant tension proportional to the volume / amount of rectal filling, with a gradual decrease in tension as the defecation process begins. The constant baseline tension of the puborectalis results in a "torsion" towards the combined anorectal transition, forming a 90° angle between the anal canal and the rectum. This anorectal angle also helps maintain anal continence.Another function of the puborectalis muscle is to retain at least partially formed stool if the external anal sphincter is damaged.

[0004] The puborectalis muscles cannot control farts or liquid stool. Anal control for these stool types is provided by the interaction of the internal and external anal sphincters. The hemorrhoidal cushions provide an airtight obstruction. In the resting state, the anal canal is blocked by the continuous tonic activity of the external anal sphincter and the baseline resting pressure of the internal anal sphincter. As a continuation and extension of the circular smooth muscle layer of the colon, the internal anal sphincter provides approximately 75-85% of the baseline pressure of the closed anal canal. The activity of this smooth muscle component is completely inhibited by rectal distension, the so-called rectoanal inhibitory reflex. This relaxation is accompanied by a reflex contraction of the external anal sphincter and puborectalis muscles to prevent defecation.

[0005] As mentioned above, current treatments are primarily aimed at correcting the tear in the sphincter through surgery. This results in short-term improvement of the symptoms mentioned above.

[0006] Mild anal incontinence can be treated with conservative methods, which can result in improvement of symptoms. However, in more severe cases, corresponding surgical interventions often result in short-term improvement with little chance of success.

[0007] Conservative treatment includes dietary changes in addition to increased fiber intake and, in cases of impaired anal sensitivity, the use of anal packs and rectal enemas. If necessary, loperamide (loperamid) combined with a bile acid binder decreases bowel motility and increases anal sphincter pressure. A newer form of therapy is the use of topically applied estrogens for postmenopausal women, but randomized studies are lacking.

[0008] However, a significant number of patients require surgical intervention: most commonly, sphincter repair (adjacent or overlapping) is performed for the acute treatment of traumatic injury following childbirth, but less frequently after anal sphincter injury from other conditions. The short-term outlook is good, but the long-term results are poor.

[0009] For stress urinary incontinence, it has been suggested that myogenic cells can be injected into the site of injury to improve stress urinary incontinence. However, stress urinary incontinence cannot be compared with anal incontinence because the etiology of the two different diseases is completely different. In addition, the two systems (urinary system vs. anal system) do not have similar functions. The urinary tract only provides adequate fluid control. The rectum is able to control solid, liquid as well as gaseous contents. This requires completely different sensory requirements. Therefore, the anatomy of the urogenital tract and the rectum is completely different. For example, while there is an external anal sphincter that surrounds the rectum, there is no equivalent that completely surrounds the urethra. In addition, there is hardly any striated muscle found on the dorsal side of the urethra in adult men, while the external anal sphincter of the rectum is a completely circular striated muscle.

[0010] Myoblasts (precursors of muscle fibers) are mononuclear muscle cells that are different from other types of cells in many ways. Myoblasts naturally fuse to form multinuclear myotubes after mitosis, resulting in long-term expression and delivery of biologically active proteins. Myoblasts have been used for gene delivery to muscles for muscle-related diseases such as Duchenne muscular dystrophy, as well as for non-muscle-related diseases, for example, gene delivery of human adenosine deaminase for adenosine deaminase deficiency; Gene transfer of human proinsulin for diabetes; Gene transfer for the expression of tyrosine hydroxylase for Parkinson's disease; Transfer and expression of factor IX for hemophilia B, delivery of human growth hormone for growth retardation.

[0011] The use of myoblasts to treat muscle degeneration, repair tissue damage or treat disease is disclosed in US Patents 5,130,141 and 5,538,722. In addition, myoblast transplantation has been used to repair myocardial dysfunction Robinson et al., 1995; Murry et al., 1996; Gojo et al., 1996; Zibaitis et al., 1994.

[0012] Myoblasts can also be used to repair muscle damage involved in the maintenance of controlled conditions, particularly urinary and / or anal incontinence.Skeletal muscle-derived cells containing myoblasts are called skeletal muscle progenitor cells, which can undergo differentiation to repair muscle damage in adults.

[0013] Several methods for isolating skeletal muscle derived cells are described in the prior art. A unique method for isolating skeletal muscle derived cells is to apply pre-plating technology. Thus, cells are obtained from muscle tissue, and their single cell suspensions are continuously transferred to different cell culture containers, also known as pre-plating, to remove non-myogenic cells (such as fibroblasts) and enrich myogenic cells (such as myoblasts). For example, Rando and Blau, 1994 describes such purification of myoblasts in cell culture (TA Rando and HMBlau, "Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.," J. Cell Biol., vol. 125, no. 6, pp. 1275–1287, Jun. 1994). Compared with a single pre-plating, the use of multiple pre-plating steps improves the purity and survival of myoblasts, which is essential for the therapeutic effect of myoblast transplantation therapy (Z. Qu, L. Balkir, JCT van Deutekom, PD Robbins, R. Pruchnic, and J. Huard, "Development of Approaches to Improve Cell Survival in Myoblast Transfer Therapy," J. Cell Biol., vol. 142, no. 5, pp. 1257–1267, Sep. 1998).

[0014] The disadvantage of the method based on pre-plating technology is that the performance of several pre-plating steps is time-consuming. Although cells are passaged multiple times by pre-plating, in order to achieve the desired number of cells with effective purity, further re-plating is required (B. Gharaibeh et al., "Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique," Nat. Protoc., vol. 3, no. 9, p. 1501, Aug. 2008; A. Lu et al., "Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics," Gene Ther., vol. 15, no. 15, pp. 1116–1125, May 2008). Therefore, during pre-plating and re-plating, the extended time of culturing and increasing the number of myoblast doublings can lead to senescence, accompanied by growth arrest, and even failure to reach the desired cell number for myoblast transplantation (WE Wright and JW Shay, "Historical claims and current interpretations of replicative aging," Nat. Biotechnol., vol. 20, no. 7, pp. 682–688, Jul. 2002). In addition, even if improved purity can be achieved, the lack of sensitivity of pre-plating may also lead to a reduction in the total number of myoblasts (BC Syverud, JD Lee, KW Van Dusen, and LM Larkin, "Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering," J. Regen. Med., vol. 3, no. 2, 2014).

[0015] Other methods for isolating myoblasts, such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), appear promising, but are not optimal for myoblast isolation due to potential damage to cells from the high-voltage laser intensities applied in FACS and concerns about the retention of magnetic antibodies used in MACS.

[0016] In view of these shortcomings of prior art methods, new methods for providing skeletal muscle derived cells (SMDCs) are needed.

[0017] This object is solved by the subject matter defined in the claims.

[0018] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0019] Figure 1 Phase contrast micrographs (100X magnification) showing SMDC adhered to a standard cell culture flask.

[0020] Figure 2 A is a FACS analysis showing purity and viability of SMDC. Histogram A is a CD56 analysis and shows that 99.3% of the cells are positive for CD56. Histogram B shows a viability analysis of 7AAD and shows that 98.77% of the cells are alive.

[0021] Figure 3 A 100X magnified micrograph showing the purity of SMDCs as demonstrated by immunostaining for the myogenic marker desmin. Positive cells appear darker stained, while negative cells are unstained.

[0022] Figure 4 Photomicrograph showing SMDC induced to differentiate into multinucleated myotubes. Myotubes express myogenic desmin as shown by dark staining.

[0023] Figure 5 FACS analysis of A2B5 staining of SMDC is shown. Isotype control staining is shown in histogram A, compared to A2B5 antibody staining, which is shown in histogram B. Histogram B shows that 96% of the cells are positive for A2B5.

[0024] Figure 6 The gene expression of A2B5-reactive antigen synthetase is shown. The average read counts per gene annotation of ST3GAL1, ST3GAL2 and ST3GAL3 show that the expression of these mRNAs in the SMDC obtained by the present invention is higher (ST3GAL1: 17.72; ST3GAL2: 20.91; ST3GAL3: 205.89) compared with the commercially available SMDC (ST3GAL1: 5.97; ST3GAL2: 9.59; ST3GAL3: 97.81).

[0025] Figure 7Comparison of %CD56 positive cells in cells obtained from Example 1 (meaning with a cooling step), or Example 9 (meaning without a cooling step) is shown. Cells obtained from Example 1 were shown to contain significantly (p<0.05 in student's t-test; *) higher %CD56 positive cells (mean±SD: 93.28±8.37) compared to cells obtained from Example 9 (mean±SD: 77.11±8.37). Data are shown as mean±SD of cells derived from muscle biopsies of three separate human donors, which were split in half to perform procedures according to Examples 1 and 9 on cells from the same donor.

[0026] Figure 8 FACS analysis showing expression of the mesenchymal marker CD105 on SMDCs is shown. Histogram B is a CD105 analysis and shows that 98.69% of SMDCs are positive for CD105. Histogram A shows accurate setting of the positive threshold by showing that the isotype control is positive in only 0.31% of SMDCs.

[0027] Fig. 9 The AChE activity (mU) between CD56+ SMDC (containing approximately 100% of cells expressing CD56) and CD56- SMDC (containing approximately 30% of CD56-positive cells) was shown. rel The neuromuscular regeneration capacity of SMDC according to the present invention was determined by PCR (100 μg protein per 100 μg protein). AChE activity was measured as described in Example 11. CD56+SMDC showed significantly (p<0.05 in student's t-test; *) higher AChE activity than CD56-SMDC. Data are shown as mean ± SD of cells derived from muscle biopsies of three separate human donors.

[0028] Fig.10 The neuromuscular regenerative capacity of SMDC according to the present invention is shown by comparing the fusion capacity (% fusion index) between CD56+SMDC (containing about 100% cells expressing CD56) and CD56-SMDC (containing about 30% CD56 positive cells). Fusion capacity quantification was performed as described in Example 12. CD56+SMDC exhibited a significantly (p<0.05 in student's t-test; *) higher % fusion index compared to CD56-SMDC. Data are shown as mean ± SEM of cells derived from muscle biopsies of three separate human donors.

[0029] When used in conjunction with the term "comprising" in the claims and / or description, the use of the words "a" or "an" may mean "one", but is also consistent with the meaning of "one or more", "at least one" and "more than one".

[0030] The term "about" means the stated value, plus or minus 5% of the stated value, or taking into account the standard error of measurement for the given value.

[0031] As used herein, the term "anal incontinence" refers to any unwanted loss of intestinal contents (such as farts, liquid or solid stool) through the anus. The term encompasses all three severity levels: Grade 1 = gas only, Grade 2 = liquid and soft stools, Grade 3 = solid formed stools.

[0032] As used herein, the term "anal sphincter" or "anal sphincter organ" refers specifically to the external anal sphincter and the puborectalis muscle, which is part of the levator ani muscle. But it also includes the pubococcygeus, coccygeus, iliococcygeus, and pudendal nerve.

[0033] The term "skeletal muscle derived cells" or "SMDC" refers to multinuclear fusion competent cells (e.g., myoblasts), which may be primary cells and / or cells cultured in vitro, and or other cells with myogenic potential (e.g., from liposuction tissue or other tissues with stem cells such as bone marrow). The term also includes cells derived from fat, which can be separated and used for the culture of skeletal muscle cells. The term "skeletal muscle derived cells" or "SMDC" also refers to a cell population isolated from muscle tissue. Typically, such a cell population includes other cells that do not have myogenic potential. Such cells are referred to herein as "non-myogenic cells" or "skeletal muscle derived non-myogenic cells", and are preferably CD56 negative and / or desmin negative. Therefore, as used herein, the term "skeletal muscle derived cells" or "SMDC" preferably refers to a cell population comprising at least 30, 40, 50, 60, 70, 80, 90, 95, 98 or 100% of multinuclear fusion competent cells.

[0034] As used herein, the term "penetration" refers to the process of introducing an injection device (eg, a needle) into human tissue without interfering with the injection process.

[0035] As used herein, the term "injection" refers to the discharge of an injection solution containing the above-mentioned cells from an injection device into a specific part of the human body, particularly into or near the muscle tissue that provides anal control. The injection process can be, but is not limited to, static, that is, the injection device remains at the achieved position. Alternatively, the injection process is dynamic. For example, in some embodiments of the present invention, the injection occurs simultaneously with the withdrawal of the injection device from the injection site.

[0036] As used herein, the term "injection site" refers to the site in the human body, such as near or near the muscle tissue that provides anal control, where the injection process begins. The injection site need not be the same site where the injection process ends.

[0037] As used herein, the term "injection device" refers to any device suitable for penetrating human tissue to reach an injection site of interest and capable of delivering a solution, particularly a solution comprising myogenic cells, to the injection site of interest.

[0038] As used herein, the term "fecal incontinence" refers simply to the undesired loss of liquid or formed stool through the anus.

[0039] As used herein, the term "passive incontinence" refers to the lack of sensory recognition of the loss of feces. This includes low anal baseline pressure values ​​and lack of sensory ability of the anal and rectal mucosa.

[0040] As used herein, "imperative defecation" or "imperative urgency" refers to a person's lack of ability to delay defecation for more than five minutes. Such patients must go to the bathroom immediately.

[0041] As used herein, the term "CD56+" or "CD56 positive" refers to cells expressing the cell marker CD56. The term "CD56+" or "CD56 positive" can also be used for cell populations comprising different cell types if preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cell population expresses the cell marker CD56.

[0042] As used herein, the term "CD56-" or "CD56 negative" refers to cells that do not express the cell marker CD56. The term "CD56-" or "CD56 negative" may also be used for cell populations comprising different cell types if preferably at most 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% or 0% of the cell population expresses the cell marker CD56.

[0043] As used herein, the term "A2B5+" or "A2B5 positive" refers to cells expressing the cell marker A2B5. The term "A2B5+" or "A2B5 positive" can also be used for cell populations comprising different cell types if preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cell population expresses the cell marker A2B5.

[0044] As used herein, the term "A2B5-" or "A2B5 negative" refers to cells that do not express the cell marker A2B5. The term "A2B5-" or "A2B5 negative" can also be used for cell populations comprising different cell types if preferably at most 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% or 0% of the cell population expresses the cell marker A2B5.

[0045] As used herein, the term "desmin positive" refers to cells that express the cell marker desmin. The term "desmin positive" can also be used for cell populations comprising different cell types if preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cell population expresses the cell marker desmin.

[0046] As used herein, the term "desmin negative" refers to cells that do not express the cell marker desmin. The term "desmin negative" may also be used for cell populations comprising different cell types if preferably at most 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% or 0% of the cell population expresses the cell marker desmin.

[0047] As used herein, the term "CD105+" or "CD105 positive" refers to cells expressing the cell marker CD 105. The term "CD105+" or "CD105 positive" can also be used for cell populations comprising different cell types if preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cell population expresses the cell marker CD105.

[0048] As used herein, the term "CD105-" or "CD105 negative" refers to cells that do not express the cell marker CD 105. The term "CD105-" or "CD105 negative" can also be used for cell populations comprising different cell types if preferably at most 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% or 0% of the cell population expresses the cell marker CD105.

[0049] As used herein, the term "differentiation medium" refers to a cell culture medium that induces fusion of multinuclear fusion competent cells or myogenic cells (e.g., myoblasts). However, the term also refers to a cell culture medium that does not contain any substance necessary to induce fusion, so that multinuclear fusion competent cells or myogenic cells can fuse without respective induction.

[0050] As used herein, the term "cell growth medium" refers to any medium suitable for the incubation of mammalian cells (eg, SMDCs) that allows the mammalian cells to attach to the surface of a culture vessel.

[0051] According to the present invention, a method for obtaining skeletal muscle derived cells (SMDC) is provided.

[0052] The first subject of the present invention relates to a method for obtaining skeletal muscle derived cells (SMDC), which comprises the following steps: (a) cooling a sample obtained from skeletal muscle tissue in a buffer; (b) processing and cooling the sample; (c) resuspending the sample of step (b) in a culture medium with serum containing at least one enzyme and heating to 38°C for 1 to 20 hours; precipitating the sample, and (d) resuspending the precipitate of the sample of step (c) to provide a single cell suspension of the sample of step (c), thereby obtaining SMDC.

[0053] The inventors have found that carrying out the method of the present invention advantageously allows obtaining and enriching SMDCs that exhibit high myogenic capacity without the need for pre-plating steps. As measured, for example, by a suitable potency assay, these enriched SMDCs are highly pure for myogenic markers such as CD56 and desmin, indicating that myogenic potential increases. In addition, since the cells of the present invention must undergo less subculture, it is advantageous to enrich SMDCs before the initial plating, resulting in a lower number of senescent cells and high viability. In addition, the present invention not only generates cells expressing myogenic markers such as CD56 and desmin, but also expresses markers of neural precursor cells such as A2B5, which also indicates the potential for neuromuscular support. In fact, the SMDCs obtained by the present invention differ from the cells of the prior art in the gene expression of A2B5 reactive antigen synthases ST3GAL1ST3GAL2 and ST3GAL3 (which are necessary for A2B5 reactive antigen neuromuscular support). In summary, the enriched SMDC obtained by the present invention has high purity and viability, and is therefore suggested to have high clinical efficacy in supporting neuromuscular connections and regenerating muscle weakness, particularly in cases of urinary and / or fecal incontinence.

[0054] In a preferred embodiment of the invention, step (a) comprises performing a muscle biopsy.

[0055] Such muscle biopsies as a source of myogenic cells can be obtained from the muscle at the site of injury or from another area more accessible to the clinical surgeon. The site of the biopsy is not limited to a unique skeletal muscle and can be, for example, from the upper arm. The size of the biopsy can contain approximately 1 cm x 1 cm x 1 cm or larger.

[0056] For the use of myoblasts to treat muscle damage, such as for the treatment of incontinence, the myoblasts are preferably isolated from a skeletal muscle biopsy of the subject to be treated.

[0057] In a further preferred embodiment, step (a) is carried out at a temperature below 16°C, preferably at a temperature in the range of 1 to 16°C, preferably 4 to 10°C, particularly preferably at 7°C; and over a time range of up to 96 hours.

[0058] Therefore, it is preferred that the process of the present invention is carried out in step (a) at a temperature range of 1 to 16° C. or at any temperature between this range, such as at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15° C. or any intermediate temperature within this range. In a further preferred embodiment of the present invention, the temperature range is from 6 to 8° C. In a further preferred embodiment, the temperature range is below 4° C., preferably in the range of 1 to 3° C.

[0059] Furthermore, it is preferred that the process of the present invention is carried out in step (a) over a time range of up to 96 hours, or any time within this range, such as 12 to 96 hours, 12 to 72 hours, 12 to 48 hours, 24 to 96 hours, 24 to 72 hours, 24 to 48 hours or any other intermediate range.

[0060] Preferably, step (b) comprises the use of scissors, a scalpel, forceps, a filter or a ball mill and a centrifuge. However, it will be apparent to a person skilled in the art that any other means allowing mincing or chopping of skeletal muscle tissue is contemplated according to the present invention.

[0061] In a further preferred embodiment, step (b) is carried out at a temperature in the range of 1 to 16°C or at any temperature between this range, such as at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15°C or any intermediate temperature within this range. In a further preferred embodiment of the present invention, step (b) is carried out at a temperature in the range of 4 to 8°C, particularly preferably 4°C. In a further preferred embodiment, the temperature range is below 4°C, preferably in the range of 1 to 3°C.

[0062] Further preferably, step (b) of the present invention is carried out for different time ranges, such as 2 to 48 hours, or any time within this range, such as 2 to 36 hours, 2 to 24 hours, or any other intermediate range.

[0063] Therefore, it is foreseeable that step (b) of the method of the present invention is carried out by processing skeletal muscle tissue and cooling the processed skeletal muscle tissue for different time periods. In a preferred embodiment of the present invention, it is foreseeable that cooling of the sample is carried out after the processing step. The inventors have found that cooling the processed sample is advantageous because it leads to a critical reduction in non-myogenic cells such as fibroblasts, which is achieved because non-myogenic cells such as fibroblasts are sensitive to temperature (because they have higher metabolic activity than resting myogenic cells). Due to this characteristic, non-myogenic cells such as fibroblasts die under low temperature conditions. Therefore, the cooling in step (b) of the method of the present invention provides an enrichment of myogenic cells. This enrichment can be achieved without any pre-laying steps. Therefore, the method of the present invention provides a method for enriching and purifying myogenic cells well without time-consuming and cost-consuming steps.

[0064] The importance of the cooling step is demonstrated in the following examples in the form of comparative data. The number of CD56 positive cells, which is shown to increase significantly. CD56 positive cell representation indicates a marker of the purity of the SMDC obtained. Therefore, the method of the present invention represents a method for obtaining a higher quantity and higher quality SDMC compared to the prior art method of not carrying out the cooling step as foreseen in the method of the present invention.

[0065] CD56, also known as neural cell adhesion molecule (NCAM), is a myogenic commitment marker expressed in skeletal muscle myoblasts in vitro (Belles-Isles et al., 1993) and smooth muscle tissue in vivo (Romanska et al., 1996). CD56 is present in fusion-active desmin+SMDC. In particular, it has been shown that CD56 marks a population of SMDCs that are capable of forming multinucleated myotubes and express higher enzyme acetylcholinesterase (AChE) activity than CD56-negative SMDCs (Thurner et al., 2018). The high AChE activity of SMDCs used to treat patients with fecal incontinence is in fact associated with a high therapeutic success rate in reducing symptoms of fecal incontinence (Thurner et al., 2018). Therefore, for patients with successful treatment of neuromyopathy and / or myopathy such as fecal incontinence, SMDCs with high purity for CD56 and therefore high AChE activity are desired. The present invention provides a method for isolating SMDCs with high purity for CD56 and high AChE activity.

[0066] Preferably, it is foreseen that step (c) comprises enzymatic treatment with a solution comprising any one or more selected from the group consisting of trypsin, papain, elastase, hyaluronidase, collagenase, deoxyribonuclease and DNase. In a preferred embodiment, step (c) foresights the use of collagenase.

[0067] According to the present invention, it is preferably foreseen that the resuspension in step (c) comprises the centrifugation of the sample of step (b), discarding the supernatant and resuspending the cell pellet in a culture medium with serum comprising at least one enzyme. In addition, in a preferred embodiment of the present invention, it is also preferred that in step (c) one or more washing steps are performed on the cells, comprising vortexing the cells in a suitable solution such as a buffer. According to the present invention, after the incubation time, the resuspended sample of step (b) in a culture medium with serum comprising at least one enzyme is centrifuged to precipitate the cells of the sample and discard the enzyme-containing supernatant.

[0068] In a particularly preferred embodiment of the invention, the enzyme in step (c) is trypsin.

[0069] In a further preferred embodiment of the present invention, step (c) is carried out at a temperature in the range of 25 to 38°C, preferably 36 to 38°C, particularly preferably at 37°C.

[0070] Preferably, step (d) preferably comprises at least one method selected from the group consisting of: FACS sorting, centrifugation, electrokinetic sorting, acoustophoresis sorting, bead-based cell sorting, and optical sorting.

[0071] Methods for obtaining single cell suspensions are well known in the art. An example of a suitable enrichment method is magnetic activated cell sorting The MACS method allows cell separation by incubating cells with particles (http: / / en.wikipedia.org / wiki / Magnetic_nanoparticles) coated with antibodies against specific surface antigens. Subsequently, the incubated cells are transferred to a column placed in a magnetic field. In this step, cells that express the antigen and are therefore attached to the nanoparticles remain on the column, while other cells that do not express the antigen flow through the column. In this way, cells can be separated positively and / or negatively with respect to specific antigens. Another example of a suitable enrichment method is fluorescence activated cell sorting. As described by Webster et al. (Exp Cell Res. 1988 Jan; 174(1): 252-65).

[0072] In a further preferred embodiment of the present invention, a further optional step (e) is carried out after step (d), which comprises incubating the single cell suspension obtained in step (d), wherein the incubation in step (e) is preferably carried out at a temperature in the range of 25 to 38°C, preferably 36 to 38°C, particularly preferably at 37°C, thereby obtaining adherent SMDCs.

[0073] This further optional incubation step allows the cells to grow to obtain a higher number of SMDCs. Those skilled in the art will adjust the culture conditions accordingly, such as culture medium and temperature, to obtain the maximum amount of SMDCs.

[0074] Further preferably, it is foreseen that step (e) is optionally followed by a further step (f) comprising discarding the non-adherent cells of step (e), wherein step (f) is preferably performed after at least 6 hours to 4 days.

[0075] According to the present invention, this optional step preferably allows the discarding of non-myogenic cells or cells floating in the culture vessel, such as dead cells. Thereby further enriching the desired adherent cells. A person skilled in the art will carry out the optional step (f) at an appropriate time within the preferred range of 6 hours to 3 days according to the actual need for carrying out such steps. Whether such optional step (f) is indeed needed can be defined, for example, by observing the cells under a microscope and determining the number of non-adherent cells in the cell culture.

[0076] In a further preferred embodiment of the present invention, step (f) is optionally followed by a further step (g) of propagating the adherent cells of step (e), the propagation in step (h) comprising culturing the adherent cells to 70 to 80% confluence for 1 to 5 generations.

[0077] In light of the present invention, one skilled in the art will be able to determine the necessary duration and number of passages to achieve the desired 70 to 80% confluence.

[0078] A second subject matter of the present invention relates to SMDCs, wherein the SMDCs comprise at least 60% CD56 positive and 60% A2B5 positive cells.

[0079] In a further preferred embodiment of the present invention, the SMDC is CD105 positive, which means that the SMDC expresses CD105 on its cell surface.

[0080] In a preferred embodiment of the invention, at least 60%, 70%, 80%, 90%, 95% or 98% or any intermediate range between said values ​​of SMDCs are CD105 positive, meaning that SMDCs express CD105 on their cell surface.

[0081] CD105 is also known as endoglin. It is a type I integral membrane homodimer protein with a subunit of 90kD, which is in vascular endothelial cells and placental syncytiotrophoblast cells. CD105 is weakly expressed on stromal fibroblasts. It is also expressed on activated monocytes and tissue macrophages. CD105 is expressed in activated endothelial cells in tissues undergoing angiogenesis such as tumors, or in the case of wound healing or skin inflammation. CD105 is a component of the TGF-β receptor system in human umbilical vein endothelial cells, and binds to TGF-β1 and β3 with high affinity, but not to TGF-β2. It has been shown that TGF-β receptors are necessary for skeletal muscle differentiation. In fact, it has been shown that TGF-β receptors are necessary for the fusion ability of myogenin, skeletal muscle differentiation factor and myoblasts. Therefore, CD105 positive cells can be preferably used for SMDC and SMDC obtained by the method disclosed in the present invention.

[0082] In a further preferred embodiment of the present invention, the SMDC is desmin positive, which means that the SMDC expresses desmin on its cell surface.

[0083] In a preferred embodiment of the invention, at least 60%, 70%, 80%, 90%, 95% or 98% or any intermediate range between said SMDC values ​​are desmin positive, meaning that the SMDC express desmin on their cell surface.

[0084] In a further preferred embodiment of the present invention, the SMDC is Pax7 positive, which means that the SMDC expresses Pax7 on its cell surface.

[0085] In a further preferred embodiment of the present invention, the SMDC is Myf5 positive, which means that the SMDC expresses Myf5 on its cell surface.

[0086] A2B5 reactive antigens belong to the c-series ganglioside family, the most abundant glycolipid family in the nervous system, where they exhibit functions such as cell-cell adhesion, recognition, and signal transduction. Loss of gangliosides leads to severe neurological defects, such as motor neuron regeneration defects in c-series ganglioside-deficient mice (RK Yu, Y.-T. Tsai, T. Ariga, and M. Yanagisawa, "Structures, biosynthesis, and functions of gangliosides—An overview," J. Oleo Sci., vol. 60, no. 10, pp. 537–544, 2011). Gangliosides are also found in large quantities at the neuromuscular junction (NMJ). Treatment of NMJ with anti-ganglioside antibodies leads to severe defects in NMJ, supporting the view that gangliosides are essential in NMJ (JJ Plomp and HJ Willison, "Pathophysiological actions of neuropathy-related anti-ganglioside antibodies at the neuromuscular junction," J. Physiol., vol. 587, no. Pt 16, pp. 3979–3999, Aug. 2009). In addition, gangliosides specifically mark stem cells that are thought to have regenerative potential (RK Yu, Y.-T. Tsai, T. Ariga, and M. Yanagisawa, "Structures, biosynthesis, and functions of gangliosides—An overview," J. Oleo Sci., vol. 60, no. 10, pp. 537–544, 2011). A2B5-reactive gangliosides are synthesized by sialyltransferases. Among them, ST3GAL1 and ST3GAL2 are highly expressed in the brain and have the highest in vitro glycosphingolipid receptor activity (ERSturgill et al., "Biosynthesis of the majorbrain gangliosides GD1a and GT1b," Glycobiology, vol. 22, no. 10, pp. 1289-1301, Oct. 2012). The mRNAs of both ST3GAL1 and ST3GAL2 are expressed in the SMDC obtained by the present invention, and the expression levels are greater than those of commercially available SMDC.Taken together, SMDCs with myogenic regenerative capacity and simultaneous expression of A2B5-reactive antigens can not only regenerate skeletal muscle but also provide neuromuscular support during muscle regeneration, especially at newly formed neuromuscular junctions.

[0087] SMDCs that can be used to treat muscle dysfunction, in particular to treat incontinence such as urinary incontinence and / or anal incontinence, preferably exhibit a characteristic expression pattern. Preferably, more than about 60%, 70%, 80%, 90%, 95% or 98% of the SMDCs express CD56 and A2B5. Preferably, the SMDCs do not express CD34, Sca-1 and MyoD. Therefore, the term "not expressing" means that preferably less than 40%, 30%, 20%, 10%, 5% or 2% of the SMDCs express the markers. The expression pattern of SMDCs as described above can be used to determine the myogenic index of cell cultures without differentiation requirements. Therefore, the expression pattern of the SMDCs can be used to verify whether skeletal muscle-derived cells can be used to treat muscle dysfunction, in particular for the treatment of incontinence such as urinary incontinence and / or anal incontinence.

[0088] Another subject of the invention relates to the SMDC obtained according to the process of the invention.

[0089] According to the present invention, in view of the different expression of A2B5 markers and / or A2B5 marker reactive antigen synthases, SMDC obtained by the method of the present invention can be clearly distinguished from cells of the prior art. Since A2B5 reactive gangliosides are synthesized by sialyltransferases, these sialyltransferases can be used as indirect markers of A2B5 expression. Among the three important sialyltransferases ST3GAL1, ST3GAL2 and ST3GAL3, ST3GAL1 and ST3GAL2 are highly expressed in the brain and have the highest in vitro glycosphingolipid receptor activity (ERSturgill et al., "Biosynthesis of themajor brain gangliosides GD1a and GT1b," Glycobiology, vol.22, no.10, pp.1289-1301, Oct.2012). Detecting the expression of ST3GAL1 and ST3GAL2 allows indirect proof of the expression of A2B5. Therefore, the novel process of the present invention allows obtaining different SMDCs compared to those obtained with processes known in the art.

[0090] In a further preferred embodiment of the present invention, SMDC and SMDC obtained by the method according to the present invention are characterized by the expression of different markers. According to the present invention, it is foreseeable that SMDC is characterized by the positive expression of one, two, three, four or five of the markers CD56, A2B5, CD105, desmin, Myf5 and Pax7. Further preferably, it is foreseeable that SMDC and SMDC obtained by the method according to the present invention are characterized by the expression of different marker combinations, and the different marker combinations are selected from the positive expression of at least one or more markers selected from CD56, A2B5, CD105, Myf5 and Pax7, and the negative expression of at least one or more markers selected from CD34 and MyoD. Preferably, for any one or more of CD56, A2B5, CD105, Myf5 and Pax7, at least 60%, 70%, 80%, 90%, 95% or 98% of SMDC or any intermediate range between the values ​​are positive, which means that SMDC expresses CD56, A2B5, CD105, Myf5 and / or Pax7 on its cell surface. Further preferably, at most 59%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% or 0% of the cell population express any of CD34 and / or MyoD.

[0091] In a particularly preferred embodiment of the invention, the SMDC and the SMDC obtained by the method according to the invention are characterized by a positive expression of CD56, A2B5, CD105, Myf5, Pax7 and a negative expression of CD34 and MyoD.

[0092] Another subject of the present invention relates to the SMDC obtained according to the process of the invention for use as a pharmaceutical composition.

[0093] According to the present invention, the SMDC obtained according to the method of the present invention can be used as an active ingredient in a pharmaceutical composition.

[0094] Another subject of the invention relates to SMDC obtained according to the method of the invention for use in a method for improving neuromuscular connections.

[0095] Preferably, SMDC according to the present invention is used in a method for improving the prevention and / or treatment of neuromyopathy and / or myopathy. Preferably, SMDC is used in a method for preventing and / or treating neuromyopathy and / or myopathy such as fecal incontinence and / or urinary incontinence.

[0096] According to the present invention, SMDC can be used in methods that preferably foresee injection of SMDC into a subject suffering from incontinence. Typically, injection of SMDC into a given tissue or injury site comprises a therapeutically effective amount of cells in solution or suspension, preferably about 1×10 cells per 100 μl of injection solution. 5 About 6×10 6 The injection solution is a physiologically acceptable medium with or without autologous serum. The physiologically acceptable medium can be a non-limiting example of physiological saline or phosphate buffered solution.

[0097] Finally, another subject of the present invention relates to the SMDC obtained according to the process of the invention for use in a method for the treatment of muscle dysfunctions, wherein the muscle dysfunction is incontinence, in particular urinary incontinence and / or anal incontinence.

[0098] In principle, any type of anal incontinence can be treated, since strengthening the anal musculature provides better control of rectal filling. However, anal incontinence caused by perineal rupture, especially when the anal sphincter system and / or puborectalis muscles are damaged and injured, can be treated. Such perineal rupture can be caused by a wide variety of reasons as described above. However, the causes of such perineal rupture are not limited to the use of the method and SMDC of the present invention. If the patient suffers from a third or fourth degree perineal rupture, it can be treated with the method and SMDC of the present invention. This is particularly suitable for women who suffer such perineal rupture after forceps delivery, first delivery, childbirth weighing more than 4 kg, or the consequences of abnormal posture before the birth of the child. The method and SMDC of the present invention can also be used after surgical damage to the anal sphincter system and / or puborectalis muscles. In addition, if there is only transient urinary incontinence, the method and SMDC of the present invention can also be applied. Such treatment can prevent the development of complete anal incontinence. Other anal incontinence disease states treated with the method and SMDC of the present invention are passive incontinence, fecal incontinence and urge to defecate.

[0099] It should be understood that the method and SMDC according to the present invention can be used not only to treat patients who already suffer from anal incontinence, i.e., show symptoms of anal incontinence, but can also be applied to patients who do not yet suffer from anal incontinence but are at increased risk, for example, in cases where the rectal muscles have been damaged due to surgery, childbirth, accidents, etc. Another example is where the rectal muscle tissue has become thinner than in healthy individuals, or where it has degenerated due to other reasons. The method of the present invention can provide appropriate prevention to prevent the onset of anal incontinence.

[0100] The SMDC used to treat or prevent incontinence is preferably homologous to the recipient.

[0101] In a more preferred embodiment, the SMDC is autologous or allogeneic to the recipient. The SMDC can be obtained, for example, by biopsy of the recipient's biceps. Autologous SMDC reduces or minimizes the risk of allergic reactions after SMDC injection into the recipient. Preferably, SMDC is a multinuclear fusion competent cell or a myogenic cell, such as a myoblast. More preferably, the SMDC is a human cell.

[0102] Therefore, the present invention also provides a simple prevention method or treatment method for women and men suffering from urinary incontinence and / or anal incontinence or having a risk of developing urinary incontinence and / or anal incontinence, and its method is to strengthen its urethra and / or anal sphincter by using autologous skeletal muscle-derived cells. Such myogenic cell therapy allows the repair and improvement of damaged urethra and anal sphincter. According to the present invention, treatment includes needle aspiration to obtain myogenic cells, for example, and a brief follow-up treatment in which the cultured and prepared cells are injected into the patient. In addition, according to the present invention, autologous skeletal muscle-derived cells (SMDC) harvested and cultured from specific urinary incontinence and / or anal incontinence patients can be used as non-allergic agents to swell the urethra and / or rectal wall, thereby enhancing coaptation and improving the urethra and / or anal sphincter. In this aspect of the present invention, as discussed above, simple autologous muscle cell transplantation is performed.

[0103] According to the present invention, autologous skeletal muscle-derived cells administered directly into the urethral sphincter and / or anal sphincter exhibit long-term survival. Thus, autologous myoblast injection results in safe and non-immunogenic long-term survival of muscle fibers in the urethral and / or anal sphincter.

[0104] In a specific embodiment according to the present invention, about 50 to about 200 μl of a skeletal muscle-derived cell suspension (at a concentration of about 1×10 per 100 μl of injection solution) is added to the mixture. 5 About 6×10 6 The injection device can be connected to a container containing the cell suspension to be injected. For the treatment of anal incontinence, preferably about 50 μl to about 1 ml, more preferably about 0.5 ml of skeletal muscle-derived cell suspension (concentration of about 1×10 cells per 100 μl) is used. 5 About 6×10 6 The injection device can be connected to a container containing the cell suspension to be injected.

[0105] In another embodiment, the injecting step may include several individual injections, such as about 20 to about 40 injections of the skeletal muscle derived cell suspension, wherein in each injection, about 50 to about 500 μl of the skeletal muscle derived cell suspension is injected, and wherein each injection is applied to another area of ​​the anal sphincter. However, these parameters must be considered merely exemplary, and a skilled artisan will be able to readily adapt these protocols to the treatment requirements of each individual patient.

[0106] In another embodiment of the invention, the movement of the injection device towards the urethra and / or anal sphincter is monitored by ultrasound and / or EMG (electromyography) means. In a specific embodiment, a transrectal probe is introduced and the position of the transrectal probe is optimally adjusted for treating the urethra and / or anal sphincter with the method according to the invention. In another specific embodiment, skeletal muscle-derived cells are implanted in the area surrounding the urethra and / or anal sphincter defect and / or in particular in the area of ​​the urethra and / or anal sphincter defect. According to the invention, the patient can start physical exercise the day after the injection of the cells to further treat urinary incontinence and / or anal incontinence.

[0107] In another embodiment, treatment is repeated. Treatment can be repeated, for example within one year after the last treatment, after 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month after the last treatment, or within 1 to 8 weeks after the last treatment, preferably 2 to 3 weeks, or 10 to 20 days. In particular, treatment is repeated within 2-3 weeks after the last treatment with cells from the same cell culture used for previous treatment. This method allows the reduction of the injection volume per injection, and gives cells more time to adapt, integrate and strengthen muscles. In even more specific embodiments, the injection is repeated every 2 to 3 weeks until urine and / or anal incontinence is improved.

[0108] As mentioned above, one particular penetration route is through the patient's skin parallel to the course of the rectum. However, it is also believed that penetration can occur directly damaging the muscles in the vicinity of the rectum. In particular, the penetration and injection process is monitored via ultrasound imaging means. In addition, another penetration route is also considered for women, namely vaginal injection. In this case, the injection device is moved forward through the vaginal wall until the desired injection site is reached. In particular, in this case, the penetration and injection process is also monitored by ultrasound and / or EMG (electromyography) imaging means.

[0109] In another embodiment, the injection comprises injecting skeletal muscle-derived cells in the form of an "injection band". As used herein, an "injection band" refers to a length or portion of a length along an injection track, i.e., along a conduit formed by inserting a needle into muscle tissue to arrange cells. In other words, after injection, as the injection needle moves (particularly retracted along the injection track), the needle is withdrawn while the cells are discharged from the syringe in a continuous or intermittent manner. Such stable cell distribution provides continuous delivery of the injection solution (including cells) along the injection tube formed when the injection device / needle enters the target muscle tissue. In a specific embodiment, the diameter of the injection band or injection tube should be no greater than about 0.8 mm, because otherwise it will cause necrosis of skeletal muscle-derived cells in the center of the injection tube, resulting in harmful inflammation and other processes.

[0110] The injection device used with the method of the present invention can be any device capable of penetrating human tissue and capable of delivering a solution, particularly a solution comprising skeletal muscle derived cells, to a desired location in the organism of a subject (particularly a human subject). The injection device can include, for example, a hollow needle. The injection device can also be any type of syringe suitable for injecting skeletal muscle derived cells. In a more complex embodiment, the injection device can be, for example, an injection gun that injects a cell suspension by applying air pressure. In particular, the injection device is suitable for keyhole application and keyhole surgery, respectively.

[0111] By selecting an injection needle with a specific diameter, the number of injections per mm can be precisely predetermined. 3 The diameter of the injection needle is usually not more than 5 mm, as this can cause damage to muscle structures.

[0112] The ultrasound imaging means for monitoring the position and movement of the injection device can be implemented by any standard ultrasound imaging device known in the art. In addition to single-plane or dual-plane standard ultrasound probes, new ultrasound technologies such as 3D ultrasound or color Doppler ultrasound can also be used. As discussed above, in certain embodiments, the injection device includes an ultrasound imaging means.

[0113] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0114] The following examples illustrate the invention but are not to be considered limiting.

[0115] Example 1 - Obtaining SMDC

[0116] To obtain SMDC, a skeletal muscle biopsy is performed on the pectoralis major or biceps brachii of an incontinent patient. To obtain the biopsy, the skin is first opened with an incision approximately 1 cm long above the muscle until the fascia of the pectoralis major is reached. After opening the fascia, a 1 cm 3 The biopsy was transferred directly to biopsy transport medium pre-cooled to approximately 4°C, which consisted of Ham's F10 basal medium supplemented with gentamicin (final concentration 1-5 μg / ml). The biopsy was stored in biopsy transport medium at 1-11°C for approximately 26 hours. Next, the biopsy was transferred to a culture dish filled with 1x PBS. The muscle tissue was separated from the connective tissue using sterile forceps and a scalpel. The muscle tissue was then transferred to another culture dish filled with 1x PBS and cut into 2-3 mm sections using a scalpel. 2 sized pieces. After the additional transfer steps described above, the tissue blocks were further cut into 1 mm pieces. Finally, the blocks were transferred to a centrifuge tube filled with 1x PBS and centrifuged at 1300 rpm for 10 minutes. After centrifugation, the supernatant was removed and the muscle tissue was resuspended in 1x PBS supplemented with 8 μg / ml gentamicin. The muscle tissue suspension was then cooled to 2-8°C for 48 hours. After cooling, the muscle tissue suspension was centrifuged at 1300 rpm for 10 minutes, and then the supernatant was removed and 2.5 ml of Ham's F10 containing 1-5 mg / ml collagenase, 2-4% v / v Hepes buffer, 0.1-10% v / v fetal bovine serum and 5-10 μg / ml gentamicin digestion solution was added. The muscle tissue suspension was then incubated at 37°C, 5% CO 2 The suspension was then centrifuged at 1300 rpm for 10 minutes, the supernatant was removed, and the pellet was resuspended in Hams F10 containing 10-20% v / v FCS, 1-3 ng / ml bFGF, and 3-10 μg / ml gentamicin and plated on a cell culture flask. The SMDCs attached to the bottom of the flask were further maintained by changing the medium every 3-4 days and separated after reaching confluence, followed by subculture. Subculture was performed until 1x10 7 Up to 5x10 7 SMDCs.

[0117] Cell count was performed using a chemometec nucleocounter TMThe method uses nuclear propidium iodide staining and counts the number of cells in 0.2 ml. Before automatic counting, 100 μl of cell suspension is mixed with 200 μl of reagent A-lysis buffer (to permeabilize the cell membrane) and incubated at room temperature for 5 minutes. Then 200 μl of reagent B-stabilization buffer is added. The suspension is softened and precipitated with a nucleocasette TM Collect and finally measure.

[0118] Figure 1 The morphology of SMDC obtained by the present invention on a standard cell culture flask visualized by phase contrast microscopy is shown.

[0119] Example 2 - Flow Cytometry

[0120] Flow cytometry analysis was performed on a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, cells were harvested with trypsin for 5 min at 37°C, centrifuged at 400 rcf, and then resuspended in 1x PBS supplemented with 1% FCS. Cells at a concentration of 40,000 cells / reaction were incubated with 5 μL of anti-CD56-PE (Beckman Coulter Inc., France), isotype IgG1-PE (Beckman Coulter), isotype Alexa488 (Sigma), anti-CD105-PE (Beckman Coulter Inc., France), or A2B5-Alexa488 antibody (Millipore) at 4°C for 1.5 mL. The cells were incubated in the dark for 15 minutes in a test tube. The cells were washed with 1 mL of PBS, centrifuged at 400 rcf, and resuspended in 200 μL of 1x PBS in a 96-well round bottom plate for FACS analysis. After washing and resuspension, each reaction received 5 μL of the active dye 7-aminoactinomycin D (Beckman Coulter Inc., France) and the plate was incubated at 4 ° C for 10 minutes. With the help of Guava InCyte TM Cell events were acquired using the ELISA software v2.3. Histograms and dot plots were generated using at least 3000 events with a sample flow rate of 1.8 μL / mL. Positive staining was obtained by comparison with an isotype control set to at least 99% negative.

[0121] Example 3 - Immunocytochemistry

[0122] First, discard the supernatant of the cell culture dish and wash the cells three times with PBS. Permeabilization and fixation were performed by covering the cells with 4% formaldehyde solution (v / v; diluted in PBS) for 20 minutes at room temperature. Then, after each incubation step performed, the cells were washed three times with PBS. Afterwards, the cells were covered with 500 μl of hydroperoxide blocking solution (Thermo Fisher Scientific) and incubated at room temperature for 5 minutes. The primary antibody (desmin) at a final concentration of 40 μg pro-ml (w / v) was pipetted onto the cells and incubated for at least 90 minutes (37°C, 5% CO 2 ). The cells were then covered with 500 μl of biotin-conjugated secondary antibody (goat anti-rabbit, polyclonal, Thermo Fisher Scientific) and incubated under the same conditions as the primary antibody, but for at least 60 min. To visualize antibody binding, 500 μl of horseradish streptavidin peroxidase (Vectorlabs) had to be added at a final concentration of 2-5 μg / ml (diluted in PBS) and incubated at 37°C, 5% CO 2 After incubation for 20 minutes at 4 °C, the cells were then covered with 500 μl of chromogen solution and removed after 5 to 15 minutes. A final washing step with PBS was performed before the results could be observed. Desmin-positive cells were stained dark red by immunocytochemistry.

[0123] Example 4 - Purity of SMDC

[0124] SMDC obtained by the procedure described in Example 1 were tested for purity of the myogenic markers CD56 (NCAM) and desmin. The percentage of CD56 positive cells was measured by flow cytometry as described in Example 2. As described in Example 1, SMDC obtained by the present invention contained 99.3% CD56 positive cells ( Figure 2 A) and 98.77% viable cells ( Figure 2 B) In addition, one representative SMDC batch obtained by the method described in Example 1 was analyzed for desmin expression by immunocytochemistry as described in Example 3. Figure 4 The high purity of SMDC obtained by Example 1 was shown because most cells were positive for desmin staining ( Figure 3 ).

[0125] Example 5 - Cell differentiation

[0126] When the growth medium is replaced by serum-free differentiation medium, human myoblasts differentiate into syncytial myotubes. Skeletal muscle cell differentiation medium (Promo Cell) is supplemented with a skeletal muscle cell differentiation medium supplement pack (from which the medium has been obtained as described in the company's protocol) and 250 μl of gentamicin. To start differentiation, cells previously counted as described in Example 1 (in growth medium) are seeded into 4-well or 24-well culture dishes (60,000-480,000 cells). After the cells are attached to the culture dish (overnight), the growth medium is discarded, the differentiation medium is used once, and covered with 500 μl of differentiation medium.

[0127] Example 6 - Myogenic Potential of SMDC

[0128] SMDCs of the myogenic lineage are able to fuse and form syncytial myotubes in vitro, which are representative of skeletal muscle fibers in vivo. Therefore, the myogenic potential of SMDCs was tested by in vitro differentiation. Cells obtained as described in Example 1 were induced to differentiate as described in Example 5 to test the myogenic potential. SMDCs formed giant myotubes that were desmin positive (stained as described in Example 3), as shown in Figure 5. Figure 4 A representative batch of SMDCs is shown. The results confirm the myogenic potential of SMDCs, which is required for functional regenerating skeletal muscle tissue.

[0129] Example 7 - Gene Expression Analysis

[0130] After the cell counting also described in Example 1, the SMDC obtained in Example 1 was seeded in 6-well plates (NUNC Thermo Scientific) at a density of 500,000 cells per well, until 70% confluence, and then according to the manufacturer's instructions, total RNA was isolated using RNeasy RNA isolation kit (QIAGEN). RNA concentration and purity were evaluated by microfluidic analysis using denaturing gel electrophoresis and 2100 Agilent bioanalyzers. Sample preparation for microarray hybridization was performed as described in NuGEN Ovation PicoSL WTASystem V2 and NuGEN Encore Biotin Module manuals (NuGEN Technologies, Inc, San Carlos, CA, USA). In brief, 7.5ng of total RNA was reverse transcribed into double-stranded cDNA in a two-step process, and SPIA tag sequences were introduced. After additional bead purification, the cDNA purified by beads was amplified by SPIA amplification reaction. 4.5 μg of SPIA cDNA was fragmented, terminally biotinylated and hybridized with Affymetrix PrimeView Human Gene Expression arrays at 45°C for 16 hours in a GeneChip hybridization oven 640. The hybridization arrays were washed and stained on an Affymetrix Fluidics Station FS450, and the fluorescence signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluids and scanning functions were controlled by Affymetrix GeneChip Command Console v4.1.3 software. Sample processing was performed at Affymetrix Service Provider and Core Facility, "KFB-Center of Excellence for Fluorescent Bioanalytics" (Regensburg, Germany; www.kfb-regensburg.deThe summary probe set signals on a log2 scale were calculated using the RMA (1) algorithm using the Affymetrix GeneChip Expression Console v1.4 software. In order to compare the gene expression results of SMDC obtained by Example 1 with the gene expression results of SMDC obtained by the prior art method, the gene expression data of commercially available SMDC (Lonza) published by Abujarour et al. in 2014 (R. Abujarour et al., "Myogenic differentiation of muscular dystrophy-specific induced pluripotent stem cells for use in drug discovery," Stem Cells Transl. Med., vol. 3, no. 2, pp. 149-160, Feb. 2014) were downloaded from the database attached to the publication (ftp: / / ftp.ncbi.nlm.nih.gov / pub / geo / DATA / supplementary / series / GSE46633 / GSE46633_RAW.tar; last accession 21.07.2017). Comparison of the gene expression of SMDC obtained by Example 1 with that analyzed by Abujarour et al. in 2014 was performed by comparing the average reads per annotation of different genes using Partek Flow software according to the providing company (Partek Inc.). Therefore, a higher average read per annotation indicates a higher abundance of the annotated mRNA, indicating a higher gene expression.

[0131] Example 8 - Neuromuscular Support Features of SMDC

[0132] The neuromuscular support characteristics of SMDC obtained by using Example 1 were confirmed by positive binding of the A2B5 antibody to the cell surface of SMDC by flow cytometry (described in Example 2). Figure 5 As shown in, 96.16% of SMDC obtained by Example 1 were positive for A2B5-reactive antibodies, indicating that in addition to its myogenicity guarantee (desmin, CD56 expression), SMDC also showed neuronal characteristics. In order to compare the neuromuscular support characteristics of SMDC obtained by the present invention with myoblasts isolated by the prior art method, as described in Example 7, the gene expression of enzymes ST3GAL1 and ST3GAL2 necessary for the formation of A2B5-reactive gangliosides that showed the highest glycosphingolipid receptor activity in vitro was analyzed. Figure 5As shown in, compared with commercially available SMDC, the mRNA expression of both ST3GAL1 and ST3GAL2 in SMDC obtained by the invention proposed herein is higher. In addition, the expression of ST3GAL3 in SMDC obtained by Example 1 is also higher.

[0133] Example 9 - Obtaining cells without cooling after tissue dissection

[0134] As a comparative example, the method of the present invention was performed by omitting the cooling step. To obtain SMDC, a skeletal muscle biopsy was performed on the pectoralis major or biceps brachii of an incontinent patient. To perform the biopsy, the skin was first opened with an incision about 1 cm long above the muscle until the fascia of the pectoralis major was reached. After opening the fascia, a 1 cm 3 The biopsy was transferred directly to biopsy transport medium pre-cooled to approximately 4°C, which consisted of Ham's F10 basal medium supplemented with gentamicin (final concentration 1-5 μg / ml). The biopsy was stored in biopsy transport medium at 1-11°C for approximately 26 hours. Next, the biopsy was transferred to a culture dish filled with 1x PBS. The muscle tissue was separated from the connective tissue using sterile forceps and a scalpel. The muscle tissue was then transferred to another culture dish filled with 1x PBS and cut into 2-3 mm sections using a scalpel. 2 sized pieces. After the additional transfer steps described above, the tissue blocks were further cut into 1 mm pieces. Finally, the pieces were transferred to a centrifuge tube filled with 1x PBS and centrifuged at 1300 rpm for 10 minutes. After centrifugation, the supernatant was removed and the muscle tissue was resuspended in 1x PBS supplemented with 8 μg / ml gentamicin. The muscle tissue suspension was centrifuged at 1300 rpm for 10 minutes, and then the supernatant was removed and 2.5 ml of Ham's F10 digestion solution containing 1-5 mg / ml collagenase, 2-4% v / v Hepes buffer, 0.1-10% v / v fetal bovine serum and 5-10 μg / ml gentamicin was added. The muscle tissue suspension was then incubated at 37°C, 5% CO 2 The suspension was then centrifuged at 1300 rpm for 10 minutes, the supernatant was removed, and the pellet was resuspended in HamsF10 containing 10-20% v / v FCS, 1-3 ng / ml bFGF, and 3-10 μg / ml gentamicin and plated on a cell culture flask. The SMDCs attached to the bottom of the flask were further maintained by changing the medium every 3-4 days and separated after reaching confluence, followed by subculture. Subculture was performed until 1x10 7 Up to 5x10 7 SMDCs.

[0135] Example 10 - Expression of Mesenchymal Cell Markers by SMDC

[0136] The expression of mesenchymal markers by SMDCs obtained by using Example 1 was determined by measuring the binding of anti-CD105 antibody, anti-CD105-PE (Beckman Coulter Inc., France) to the SMDC cell surface by flow cytometry (as described in Example 2). Figure 8 As shown in , when the positive threshold was set according to the isotype control (0.31% positive), 98.69% of the SMDCs obtained by Example 1 were detected as CD105-reactive antibodies positive, indicating that SMDCs are mesenchymal cells and / or are of mesenchymal origin.

[0137] Example 11 - Measurement of Acetylcholinesterase Activity of SMDC

[0138] To measure acetylcholinesterase (AChE) activity, 200,000 SMDCs were seeded in gelatin-coated wells of 24-well plates and induced to differentiate as described in Example 5. Six days after the start of differentiation, the differentiation medium was carefully removed from the 24-well plates and immediately added with 300 μL of 0.5 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) solution (prepared in phosphate buffer, pH 7.2, with 0.1% triton X-100). After incubation in the dark at room temperature for 2 minutes, 50 μL of 5.76 mM iodinated acetylthiocholine (ATI) (prepared in distilled water) was added. The reaction contents were incubated in the dark at 30°C for 60 minutes, and then the optical density (OD) was measured at 412 nm on an Anthos Zeneth 340rt microplate reader (Biochrom Ltd., Cambridge, UK) (OD412nm). A blank reaction was also included, which consisted of all reagents except cells. The corrected OD412nm value was obtained by subtracting the blank average measurement from the average OD412nm. The relative mU AChE activity (AChE-mUrel) was calculated based on the linear equation of the standard curve. The AChE activity in mU was calculated by measuring 4-500 mU / ml of a ready-to-use 50 U / ml AChE stock solution (from Electrophorus electricus, AAT Inc., Sunnyvale, CA, USA) to obtain a standard curve. Standard dilutions were prepared in phosphate buffer (pH 7.2 with 0.1% triton X-100) and used immediately. For the AChE standard enzyme assay, 200 μL of each dilution was mixed with 300 μL of 0.5 mM DTNB and 50 μL of 5.76 mM ATI, respectively, and OD412nm was measured after 60 minutes in a 24-well plate. In order to standardize the cell type-specific protein concentration, the AChE activity of the measured cell amount was divided by the total protein content of these cells to obtain the mUrel AChE activity per g protein (AChE-mUrel / g protein). In order to analyze the total amount of protein in the cell population, aliquots of adherent cells differentiated according to Example 5 were first washed twice with PBS, then covered with PBST (0.1% Triton X-100), and then incubated at room temperature for 10 minutes. Next, the lysate was resuspended and transferred to The tubes were vortexed briefly and then centrifuged at 1200*g for 4 minutes. Finally, the clarified supernatant was transferred to a fresh Eppendorf tube and the protein concentration was determined by measuring the OD at 540 nm with an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK) using the Pierce BCA Protein Assay Kit (Thermo Scientific, MA, USA) according to the manufacturer's instructions.

[0139] Example 12 - Quantification of SMDC Fusion Capacity

[0140] The fusion capacity of SMDCs is based on their ability to form multinucleated myotubes and represents their myogenic potential. The fusion capacity was quantified as the fusion index, which represents the number of nuclei located within multinucleated myotubes (defined as cells containing at least 3 nuclei) divided by the total number of nuclei observed in the microscopic field. To determine the fusion index of SMDCs, 2 × 10 5Cells were seeded on gelatin-coated 24-well plates and induced to differentiate by switching from growth medium to skeletal muscle differentiation medium 24 hours after seeding. After 6 days of differentiation, the cells were washed twice with PBS and fixed with 4% PFA for 10 minutes. Next, the cells were washed three times with PBS and stained with 2μg / mL Hoechst3342 solution for 20 minutes. For each sample, at least three fields of view were captured during immunofluorescence imaging, and phase contrast images were overlaid to allow detection of nuclear and cell boundaries. After calculating the average of all analyzed fields of view, the fusion index of each captured field of view was calculated by dividing the number of nuclei in each field of view by the total number of nuclei. Only cells with at least 3 nuclei were considered myotubes. For statistical analysis, at least 3 populations derived from different patients were analyzed for each group of cells.

Claims

1. A method for obtaining skeletal muscle-derived cells, the method comprising the following steps: (a) Samples obtained from skeletal muscle tissue were cooled in buffer; (b) processing the sample and cooling the sample after processing, wherein the cooling is performed at a temperature in the range of 2 to 8° C. for 48 hours; (c) resuspending the sample of step (b) in a medium containing at least one enzyme and serum and heating to 25 to 38° C. for 1 to 20 hours; precipitating the sample, and (d) resuspending the pellet of the sample of step (c) to provide a suspension of single cells from the sample of step (c), thereby obtaining the skeletal muscle-derived cells.

2. The method according to claim 1, wherein step (a) is carried out at a temperature below 16°C; and over a period of up to 96 hours.

3. The process according to claim 2, wherein step (a) is carried out at a temperature of 4 to 10°C.

4. The method according to any one of claims 1 to 3, wherein step (b) comprises using scissors, a scalpel, forceps, a filter or a ball mill.

5. The process according to any one of claims 1 to 3, wherein the cooling in step (b) is carried out at a temperature in the range of 4 to 8°C.

6. The method according to any one of claims 1 to 3, wherein step (c) comprises enzymatic treatment with a solution comprising any one or more selected from the group consisting of trypsin, papain, elastase, hyaluronidase, collagenase and DNase.

7. The process according to any one of claims 1 to 3, wherein step (c) is carried out at a temperature in the range of 36 to 38°C.

8. The method according to any one of claims 1 to 3, wherein step (d) comprises at least one method selected from the group consisting of FACS sorting, centrifugation, electrokinetic sorting, acoustophoresis sorting, bead-based cell sorting and optical sorting.

9. The method according to any one of claims 1 to 3, wherein step (d) is followed by a further step (e), which comprises incubating the single cell suspension obtained in step (d), wherein the incubation in step (e) is carried out at a temperature in the range of 25 to 38°C, thereby obtaining adherent skeletal muscle-derived cells.

10. The method according to claim 9, wherein step (e) is followed by a further step (f) comprising discarding the non-adherent cells of step (e).

11. The method according to claim 10, wherein step (f) is followed by a further step (g) of multiplying the adherent cells of step (e), the multiplication in step (g) comprising culturing the adherent cells to 70 to 80% confluence for 1 to 5 generations.

12. A cell population of skeletal muscle-derived cells, wherein the cell population comprises at least 90% CD56-positive, 90% A2B5-positive, 90% CD105-positive and 90% desmin-positive cells, and less than 10% of the cell population express CD34, Sca-1 and MyoD, and wherein, The cell population of skeletal muscle-derived cells is obtained in a method according to any one of claims 1 to 11.

13. A cell population of skeletal muscle-derived cells according to claim 12 for use as a pharmaceutical composition.

14. The cell population of skeletal muscle-derived cells according to claim 12, for use in a method for preventing and / or treating a neuromyopathy, wherein the neuromyopathy is fecal incontinence and / or urinary incontinence.

15. A cell population of skeletal muscle-derived cells according to claim 12, for use in a method for treating a muscle dysfunction, wherein the muscle dysfunction is incontinence.

16. Use of the cell population of skeletal muscle-derived cells according to claim 12 for the preparation of a medicament for preventing and / or treating neuromyopathy, wherein the neuromyopathy is fecal incontinence and / or urinary incontinence.

17. Use of a cell population of skeletal muscle-derived cells according to claim 12 for the preparation of a medicament for treating muscle dysfunction, wherein the muscle dysfunction is incontinence.

18. Use of the cell population of skeletal muscle-derived cells according to claim 15 or the cell population of skeletal muscle-derived cells according to claim 17, wherein the incontinence is urinary incontinence and / or anal incontinence.

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