RU2796929C2 - Methods for obtaining muscle-derived cells - Google Patents

Abstract

FIELD: cell biology; medicine.

SUBSTANCE: invention relates in particular to a method for obtaining a population of cells derived from skeletal muscle (SMDCs), and to a population of SMDCs obtained in this way. To implement this method, the sample obtained from skeletal muscle tissue is first cooled in buffer. Then it is processed by subsequent cooling of the sample, and the cooling is performed at a temperature of 1–16°C for a period of time of 2–48 hours. Next, the sample is resuspended in serum medium containing at least one enzyme and heated to 25–38°C for 1–20 hours, precipitate the sample by centrifugation. Thereafter, the sample pellet is resuspended to obtain a single cell suspension from the sample, thereby obtaining a population of SMDCs. The resulting cell population of SMDCs has myogenic activity and fusion competence, and also contains at least 90% of CD56 positive, 90% of A2B5 positive, 90% of CD105 positive, and 90% of desmin positive cells.

EFFECT: invention allows to expand the arsenal of methods for obtaining SMDCs populations that can be used to prevent or treat neuromyopathies or myopathies, in particular urinary or anal incontinence, fecal incontinence.

18 cl, 10 dwg, 12 ex

Description

The present invention relates to methods for producing skeletal muscle-derived cells (SMDC), to the use of SMDC in a method for improving neuromuscular connectivity, and for use in the treatment of muscle dysfunction, where the muscle dysfunction is incontinence, in particular urinary incontinence and/or anal incontinence.

The ability to control bladder and bowel function is essential to our well-being as social beings. Loss of anal control leads to physical, physiological and social limitations. It is generally believed that anal incontinence affects mainly the elderly and people with disabilities, but these symptoms can occur in people of any age. The spectrum of anal incontinence, that is, loss of control of bowel contents, ranges from minor traces of feces on underwear to gas from the intestines, up to episodes of massive uncontrolled bowel movements of soft or hard feces. The reasons for this can be layered and complex. Regardless of the extremely degraded quality of life for the affected individual, impaired anal control results in a high health care cost factor that should not be underestimated. In addition, anal incontinence is the second most common cause of hospitalization in nursing homes (more common than dementia). A third of older people in nursing homes or hospitals have fecal incontinence.

Two skeletal muscles are important for voluntary control of the organs of retention: Musculus sphincter ani externus and Musculus puborectalis as part of Musculus levator ani. Most likely, the remaining muscle of Diaphragma pelvis (M. pubococcygeus, M. ischiococcygeus, M. iliococcygeus) plays a more or less auxiliary role. The external anal sphincter is maintained by N. pudendus. Both muscles, Musculus sphincter ani externus and Musculus puborectalis, can maintain a constant tone, in direct proportion to the volume/amount of filling of the rectum, which decreases with the onset of the defecation process. The constant initial tone of M. puborectalis leads to a "bend" of the anorectal junction towards the symphysis with the formation of a 90° angle between the anal canal and the rectum. This anorectal angle also helps maintain anal retention. An additional function of M. puborectalis is to retain at least partially formed feces when the external anal sphincter is damaged.

Control over the release of gases or liquid feces with M. puborectalis is not possible. Anal retention of these types of feces is provided through the interaction of the internal and external anal sphincter. Hemorrhoidal cushions provide an airtight barrier. At rest, the anal canal is occluded by the constant tonic activity of the external anal sphincters and the underlying resting pressure of the internal anal sphincter. The internal anal sphincter, which is a continuation and expansion of the circular smooth muscle layer of the colon, provides approximately 75-85% of the initial pressure of the closed anal canal. The activity of this smooth muscle component is completely inhibited by rectal distension, the so-called rectal anal inhibitory reflex. This relaxation is accompanied by a reflex contraction of the external anal sphincter and M. puborectalis to prevent defecation.

As indicated above, current treatments are mainly directed to the surgical repair of a sphincter tear. This results in a short-term improvement in symptoms as noted above.

Mild anal incontinence can be treated with conservative treatments, which may lead to improvement in symptoms. However, in more severe cases, appropriate surgery often results in short-term improvement with little chance of success.

Conservative treatments include dietary changes in addition to increased fiber intake and, in cases of impaired anal sensitivity, the use of anal swabs and rectal enemas. The use of loperamide, if necessary also in combination with bile acid binders, reduces intestinal motility and increases the pressure of the anal sphincter muscle. A newer form of therapy uses topical topical estrogen for post-menopausal women, but randomized trials are lacking.

However, a certain number of patients require surgery: most commonly, sphincter reconstruction (proximal or overlapping) is used as an emergency treatment for trauma after childbirth, but also secondary to anal sphincter injuries caused by other circumstances. At the same time, short-term prospects are good, and long-term results are bad.

For stress incontinence, it has been proposed to inject cells of muscle origin into the injured area to correct stress incontinence. However, stress incontinence is not comparable to anal incontinence because the causes of these two different conditions are quite different. In addition, these two systems (urinary and anal) also perform different functions. The urinary tract should only provide adequate control of fluids. The rectum is able to control solid, liquid as well as gaseous contents. This requires completely different sensory conditions. Therefore, the anatomy of the urogenital tract and the rectum is completely different. For example, while there is an external anal sphincter muscle that surrounds the rectum, there is no equivalent that completely surrounds the urethra. In addition, the striated muscle can hardly be found on the back of the urethra in an adult male, while the external anal sphincter of the rectum is a striated muscle that is completely round.

Myoblasts, the precursors of muscle fibers, are mononuclear muscle cells that differ from other cell types in many ways. Myoblasts naturally fuse to form post-mitotic multinucleated myotubes, resulting in sustained expression and delivery of biologically active proteins. Myoblasts are used to deliver genes to muscles in muscle diseases such as Duchenne muscular dystrophy, as well as in non-muscle related diseases, such as the delivery of human adenosine deaminase genes in adenosine deaminase deficiency syndrome; when transferring the human proinsulin gene in diabetes mellitus; when transferring a gene for the expression of tyrosine hydroxylase in Parkinson's disease; in the transfer and expression of factor IX in hemophilia B, for the delivery of human growth hormone in growth retardation.

The use of myoblasts to treat muscle degeneration, to repair tissue damage, or to treat disease is described in US Pat. Murry et al., 1996; Gojo et al., 1996; Zibaitis et al., 1994.

Myoblasts can also be used to heal muscle injuries involved in continence maintenance, in particular urinary incontinence and anal incontinence. Skeletal muscle-derived cells containing myoblasts are known as skeletal muscle progenitor cells that can undergo differentiation to heal muscle injuries in adults.

The prior art describes several methods for isolating cells derived from skeletal muscle. A separate method for isolating cells derived from skeletal muscle involves a pre-culture method. Here, cells are obtained from muscle tissue and the single cell suspension is transferred sequentially to different cell culture containers, known as preculture, to remove non-myogenic cells such as fibroblasts and enrich myogenic cells such as myoblasts. For example, Rando and Blau, 1994 describe such purification of myoblasts in cell culture (T.A. Rando and H.M. Blau, "Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy" J. Cell Biol, vol. 125, no. 6, pp. 1275-1287, Jun. 1994). The use of multiple preculture steps compared to a single preculture increases the purity and survival of myoblasts, which is necessary for the therapeutic effect of myoblast transfer therapy (Z. Qu, L. Balkir, J.C.T. van Deutekom, P.D. 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).

A disadvantage of methods based on the pre-culture technique is that it takes a long time to complete several pre-culture steps. Although the cells have gone through several preculture steps, additional reculture is necessary to obtain the required number of cells of effective purity (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). Thus, an increase in culture time and an increase in the number of myoblast cell duplications during pre- and re-culture can lead to senescence, accompanied by growth arrest and even the inability to reach the required number of cells for myoblast transfer. (W.E. Wright and J.W. Shay, "Historical claims and current interpretations of replicative aging," Nat. Biotechnol, vol. 20, no. 7, pp. 682-688, Jul. 2002). In addition, the lack of pre-culture sensitivity may lead to an overall decrease in the number of myoblasts, even though increased purity can be achieved. (B.C. Syverud, J.D. Lee, K.W. VanDusen, and L.M. Larkin, "Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering" J. Regen. Med., vol. 3, no. 2, 2014).

Other methods of myoblast isolation, such as fluorescent activated cell sorting (FACS) or magnetic field activated cell sorting (MACS) seem promising, but due to possible cell damage when high voltage laser radiation is used in FACS, as well as damage when retention of magnetic antibodies at MACS, these methods are not the best choice for isolating myoblasts.

In view of these shortcomings of the methods known in the art, new methods for obtaining skeletal muscle-derived cells (SMDCs) are needed.

This problem is solved with the help of the object defined in the claims.

The following figures form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention can be better understood with the help of one or more of these drawings in combination with a detailed description of the specific embodiments presented here.

On FIG. 1 shows a phase contrast microscopy image (100x magnification) of SMDCs attached to a standard culture flask.

On FIG. 2 shows a FACS analysis demonstrating the purity and viability of SMDC. Histogram A is a CD56 analysis and shows that 99.3% of the cells are CD56 positive. Histogram B is a 7AAD viability assay and shows that 98.77% of the cells are viable.

On FIG. 3 is a microscopic image (at 100x magnification) of SMDC purity shown by immunostaining for the myogenic marker desmin. Positive cells are darker stained, while negative cells are not stained.

On FIG. 4 shows a microscopic image of SMDCs induced to differentiate into multinucleated myotubules. Myotubules express the myogenic protein desmin as shown by dark staining.

On FIG. 5 shows FACS analysis of A2B5 staining of SMDC. Isotype control staining is shown in histogram A compared to A2B5 antibody staining shown in histogram B. Histogram B shows that 96% of the cells are A2B5 positive.

On FIG. 6 shows the gene expression of the enzymes that synthesize the A2B5 reactive antigen. Average values for ST3GAL1, ST3GAL2 and ST3GAL3 gene annotation suggest higher expression of these mRNAs in SMDCs obtained by the present invention (ST3GAL1: 17.72; ST3GAL2: 20.91; ST3GAL3: 205.89) compared to commercially available SMDCs ( ST3GAL1: 5.97; ST3GAL2: 9.59; ST3GAL3: 97.81).

On FIG. 7 shows a comparison of the % of CD56 positive cells in cells obtained either by Example 1 with a cooling step or by Example 9 without a cooling step. The cells obtained in Example 1 were shown to contain a significantly (p<0.05 (Student's t-test); *) higher % CD56-positive cells (mean ± SD): 93.28 ± 8.37 ) compared to the cells obtained in Example 9 (mean ± SD: 77.11 ± 8.37). Data are presented as the mean ± SD of cells obtained from muscle biopsies from three separate human donors, which were halved for the procedures of Examples 1 and 9 on cells from the same donors.

On FIG. 8 shows a FACS analysis demonstrating expression of the mesenchymal marker CD105 on SMDC. Histogram B is a CD105 analysis and shows that 98.69% of SMDCs are positive for CD105. Histogram A shows the fine-tuning of the positivity threshold by demonstrating isotype control positivity in only 0.31% of SMDC.

On FIG. 9 shows the neuromuscular regenerative capacity of SMDCs of the present invention by comparing AChE activity (mU _rel per gram of protein) between CD56+ SMDCs (containing about 100% CD56 expressing cells) and CD56-SMDCs (containing about 30% CD56 positive cells). Measurement of AChE activity was performed as described in Example 11. CD56+ SMDCs show significantly (p<0.05 (Student's t-test); *) higher AChE activity compared to CD56-SMDCs. Data are presented as mean ± SEM of cells obtained from muscle biopsies from three separate human donors.

On FIG. 10 shows the neuromuscular regenerative capacity of SMDCs of the invention by comparing the fusion capacity (% fusion index) between CD56+ SMDCs (containing about 100% CD56 expressing cells) and CD56-SMDCs (containing about 30% CD56 positive cells). Fusion ability was quantified as described in Example 12. CD56+ SMDCs show a significantly (p<0.05 (Student's t-test); *) higher % fusion index compared to CD56-SMDCs. Data are presented as mean ± SEM of cells obtained from muscle biopsies from three separate human donors.

The use of the word "a" or "an" when used together with the term "comprising" in the claims and/or in the description may mean "one", but it also corresponds to the meaning of "one or more", "at least one", and "one or more than one".

The term "about" means that the specified value is assumed to be plus or minus 5% of the specified value, or the standard error for measurements of that value.

As used herein, the term "anal incontinence" refers to any unwanted loss of intestinal contents through the anus, such as passing gases, liquid or solid feces. The term includes all three severity levels: Grade 1 = Gas only, Grade 2 = Liquid and soft feces, Grade 3 = Solid, formed feces.

As used herein, the term "anal sphincter" or "anal sphincter apparatus" refers specifically to Musculus sphincter ani externus and Musculus puborectalis as part of Musculus levator ani. However, it also includes M. pubococcygeus, M. ischiococcygeus, M. iliococcygeus, and N. pudendus.

The term "skeletal muscle-derived cell" or "SMDC" refers to multinucleated fused competent cells, such as, for example, myoblasts, which may be primary cells and/or in vitro cultured cells and alternative to other cells with myogenic potential (e.g., tissue from liposuction or other tissues containing stem cells, such as bone marrow). The term also includes cells derived from adipose tissue that can be isolated and used to culture skeletal muscle cells. The term "cell derived from skeletal muscle" or "SMDC" also refers to a population of cells isolated from muscle tissue. As a rule, such a cell population contains additional 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. Thus, the term "skeletal muscle-derived cell" or "SMDC" as used herein preferably refers to a population of cells containing at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% multinucleated fused competent cells.

As used herein, the term "penetration" refers to the method of introducing an injection device, such as a needle, into body tissue without affecting the injection process.

As used herein, the term "injection" refers to the expulsion of an injection solution containing the aforementioned cells from an injection device into a specific site in the human body, in particular into or near muscle tissue providing anal retention. The injection process may be static, that is, the injection device remains in the reached position, but is not limited to this. Alternatively, the injection process is dynamic. For example, in some embodiments of the present invention, the injection occurs simultaneously with the retraction of the injection device from the injection site.

As used herein, the term "injection site" refers to a site in the human body, such as close to muscle tissue that provides anal retention, or is such a tissue in which the injection process is initiated. The injection site does not have to be the same as the site where the injection process ends.

As used herein, the term "injection device" refers to any device suitable for penetrating human tissue in order to reach an injection site of interest, and capable of delivering solutions, in particular solutions containing muscle-derived cells, to the sites of interest for injection. injections.

As used herein, the term "fecal incontinence" refers only to the unwanted loss of liquid or formed feces through the anus.

As used herein, the term "passive incontinence" refers to the lack of sensory recognition of fecal loss. This includes low baseline anal pressures and a lack of sensory ability in the anal and rectal mucosa.

As used herein, "imperative defecation" or "imperative urination" refers to the inability of a subject to delay defecation for more than five minutes. Such a patient is forced to immediately go to the toilet.

As used herein, the term "CD56+" or "CD56 positive" refers to a cell that expresses the CD56 cellular marker. The terms "CD56+" or "CD56 positive" may also be used for a cell population containing different cell types, if preferably at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker. CD56.

As used herein, the term "CD56-" or "CD56-negative" refers to a cell that does not express the CD56 cell marker. The terms "CD56-" or "CD56-negative" may also be used for a cell population containing different cell types, if preferably not more than 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD56.

As used herein, the term "A2B5+" or "A2B5 positive" refers to a cell expressing the A2B5 cell marker. The terms "A2B5+" or "A2B5-positive" can also be used for a cell population containing different cell types, if preferably at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population expresses the cell marker. A2B5.

As used herein, the term "A2B5-" or "A2B5-negative" refers to a cell that does not express the A2B5 cell marker. The terms "A2B5-" or "A2B5-negative" may also be used for a cell population containing different cell types, if preferably not more than 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker A2B5.

As used herein, the term "desmin-positive" refers to a cell expressing the cellular marker desmin. The term "desmin-positive" can also be used for a cell population containing different types of cells, if, preferably, at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population expresses the cell marker desmin.

As used herein, the term "desmin-negative" refers to a cell that does not express the cellular marker desmin. The term "desmin-negative" can also be used for a cell population containing different types of cells, if, preferably, no more than 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent of the cell population expresses cellular marker desmin.

As used herein, the term "CD105+" or "CD105 positive" refers to a cell expressing the CD105 cell marker. The terms "CD105+" or "CD105 positive" can also be used for a cell population containing different cell types, if preferably at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population expresses the cell marker. CD105.

As used herein, the term "CD105-" or "CD105-negative" refers to a cell that does not express the CD105 cell marker. The terms "CD105-" or "CD105-negative" can also be used for a cell population containing different cell types, if preferably not more than 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD105.

As used herein, the term "differentiation medium" refers to a cell culture medium that induces fusion in multinucleated fused competent cells or in myogenic cells such as, for example, myoblasts. However, the term also refers to a cell culture medium that does not contain any substances necessary for the induction of fusion, in the case where multinucleated fusion-competent cells or myogenic cells are able to fuse without corresponding induction.

As used herein, the term "cell growth medium" refers to any medium suitable for the incubation of mammalian cells, such as SMDC, which allows said mammalian cells to adhere to the surface of the incubation container.

In accordance with the present invention, methods are provided for the production of skeletal muscle-derived cells (SMDCs).

The first object of the present invention is directed to a method for obtaining cells derived from skeletal muscle (SMDC), which includes the steps: (a) cooling the sample obtained from skeletal muscle tissue in a buffer; (b) sample processing and cooling; (c) resuspending the sample from step (b) in serum medium containing at least one enzyme, and heating to 38°C for 1-20 hours; sedimenting the sample by centrifugation; and (d) resuspending the pellet obtained in step (c) to obtain a single cell suspension from the sample from step (c), thereby obtaining SMDC.

The inventors have found that carrying out the method of the present invention advantageously allows the production and enrichment of SMDCs exhibiting high myogenic capacity without the need for pre-culture steps. These enriched SMDCs are highly pure for myogenic markers such as CD56 and desmin, indicating an increased myogenic potential as determined, for example, by a suitable activity assay. In addition, enrichment of SMDC cells prior to initial seeding is preferred as the cells need to be less subcultured, thus resulting in fewer old cells and higher viability due to the present invention. In addition, the present invention generates cells not only expressing myogenic markers such as CD56 and desmin, but also neuronal progenitor cell markers such as A2B5, suggesting possible neuromuscular support as well. Indeed, the SMDCs produced by the present invention differ from the cells known in the art by their gene expression of the ST3GAL1, ST3GAL2, and ST3GAL3 enzymes that synthesize the A2B5 reactive antigen required for neuromuscular support of the A2B5 reactive antigens. In summary, the enriched SMDCs prepared according to the present invention show high purity and viability, thus suggesting high clinical efficacy in maintaining the neuromuscular junction as well as in regenerating muscle weakness, especially in cases of urinary incontinence and/ or fecal incontinence.

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

Such a muscle biopsy, serving as a source of cells of muscle origin, may be obtained from the muscle at the site of injury or from another area that may be more easily accessible to the clinical surgeon. The biopsy site is not limited to a single skeletal muscle and may be, for example, the shoulder. The biopsy may be about 1 cm x 1 cm x 1 cm or larger.

For the use of myoblasts in the treatment of muscle injury, for example in the treatment of incontinence, said myoblasts are preferably isolated from a skeletal muscle biopsy of the subject being treated.

In another preferred embodiment, step (a) is carried out at a temperature below 16°C, preferably at a temperature in the range from 1 to 16°C, preferably from 4 to 10°C, in particular preferably at 7°C; and for up to 96 hours.

Accordingly, it is preferred that step (a) of the method of the invention is carried out in the temperature range from 1 to 16°C or at any temperature in this range, for example at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15°C or at any intermediate temperature in this interval. In another preferred embodiment of the present invention, the temperature range varies from 6 to 8°C. In another preferred embodiment, the temperature range is below 4°C, preferably in the range from 1 to 3°C.

Furthermore, it is preferred that step (a) of the method of the present invention is carried out in a time interval of up to 96 hours, or for any time in this interval, for example, 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 interval in between.

Preferably, step (b) involves the use of scissors, a scalpel, tweezers, a filter or ball grinder, and a centrifuge. However, one of ordinary skill in the art will appreciate that any other means that allows the skeletal muscle tissue to be shredded or shredded are within the scope of and are in accordance with the present invention.

In another preferred embodiment, step (b) is performed at a temperature in the range of 1 to 16°C or at any temperature in this range, for example at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15°C or at any intermediate temperature in this range. In another preferred embodiment of the present invention, step (b) is carried out at a temperature in the range of 4 to 8°C, particularly preferably at 4°C. In another preferred embodiment, the temperature range is below 4°C, preferably in the range from 1 to 3°C.

It is further preferred that step (b) of the present invention is carried out within a specified time interval, such as 2 to 48 hours, or for a time within this interval, such as 2 to 36 hours, 2 to 24 hours, or any other intermediate interval.

Accordingly, it is assumed that step (b) of the method of the present invention is performed by treating the skeletal muscle tissue and cooling the treated skeletal muscle tissue for a certain period of time. In a preferred embodiment of the invention, it is assumed that cooling of the sample is carried out after the processing step. The inventors have found that cooling the treated sample is beneficial because it leads to a significant reduction in non-myogenic cells such as fibroblasts, which is achieved due to the fact that non-myogenic cells such as fibroblasts are thermosensitive due to higher metabolic activity than immobile myogenic cells. Due to this property, non-myogenic cells such as fibroblasts die due to cold temperature conditions. As a consequence, cooling in step (b) of the method of the present invention provides an enrichment of myogenic cells. This enrichment is achieved without the need for any pre-culture steps. Therefore, the method of the present invention provides a good enrichment and purification of myogenic cells without the need for long and expensive steps.

The importance of the cooling step, in particular, is demonstrated in the comparative data shown in the examples below. It shows that the number of CD56-positive cells increases significantly. CD56 positive cells are a marker that demonstrates the purity of the resulting SMDCs. Therefore, the method of the present invention is a method that allows the production of SMDC in a larger quantity, as well as a higher quality, compared to the methods of the prior art, which do not carry out a cooling step, as provided in the method of the present invention.

CD56, also known as the neuronal cell adhesion molecule (NCAM), is a myogenic committed marker expressed in skeletal muscle myoblasts in vitro (Belles-Isles et al., 1993) and in smooth muscle tissue in vivo (Romanska et al., 1996) . CD56 is present in fused competent desmin + SMDC. In particular, CD56 has been shown to mark a population of SMDCs that are able to form multinucleated myotubules and exhibit higher acetylcholinesterase (AChE) enzymatic activity than CD56-negative SMDCs (Thurner et al., 2018). The high AChE activity of SMDCs used to treat patients with fecal incontinence is actually associated with high treatment success in terms of reducing the symptoms of fecal incontinence (Thurner et al., 2018). Thus, SMDCs that are highly pure for CD56 and thus have high AChE activity are desirable for the successful treatment of patients with neuromyopathies and/or myopathies such as fecal incontinence. The present invention provides a method for isolating SMDCs that are highly pure for CD56 and have high AChE activity.

Preferably, step (c) is contemplated to include subjecting the enzyme to a solution containing one or more enzymes selected from the group consisting of trypsin, papain, elastase, hyaluronidase, collagenase, deoxyribonuclease, and DNase. In a preferred embodiment, step (c) involves the use of collagenase.

In accordance with the present invention, it is preferably contemplated that resuspension in step (c) comprises centrifuging the sample from step (b), removing the supernatant, and resuspending the cell pellet in serum containing at least one enzyme. In addition, in a preferred embodiment of the present invention, it is also preferred that one or more washing steps be performed on the cells in step (c), including vigorously mixing the cells in a suitable solution, such as a buffer. According to the invention, the resuspended sample from step (b) in a serum medium containing at least one enzyme is centrifuged after the end of the incubation time to pellet the sample cells, and the supernatant containing the enzyme is discarded.

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

In another 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.

Preferably, step (d) comprises a method selected from at least one of FACS sorting, centrifugation, electrokinetic sorting, acousticophoresis sorting, microsphere cell sorting, and optical sorting.

Methods for preparing a single cell suspension are well known in the art. One example of a suitable enrichment method is magnetically activated cell sorting (MACS®). The MACS method allows cells to separate by incubating cells with particles (http://en.wikipedia.org/wiki/Magnetic_nanoparticles) coated with antibodies to a specific surface antigen. The incubated cells are then transferred to a column placed in a magnetic field. At this stage, 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 pass through the column. In this way cells can be divided into positive and/or negative with respect to a particular antigen(s). Another example of a suitable enrichment method is fluorescent activated cell sorting (FACS®) as described, for example, in Webster et al. (Exp Cell Res. 1988 Jan; 174(1):252-65).

In another preferred embodiment of the present invention, after step (d), an additional optional step (e) is performed, comprising incubation of the single cell suspension obtained in step (d), where the incubation in step (e) is preferably carried out at a temperature in the range from 25 to 38° C, preferably 36 to 38°C, particularly preferably at 37°C, thus obtaining attached SMDCs.

This additional possible incubation step allows the cells to grow to achieve more SMDC. One skilled in the art will adjust culture conditions such as medium and temperature, respectively, to obtain the maximum amount of SMDC.

It is also preferably contemplated that after step (e) an additional step (e) can be performed, comprising the removal of non-adherent cells from step (e), where step (e) is preferably performed after a period of at least 6 hours to 4 days.

According to the present invention, this optional step preferably allows the removal of non-myogenic cells or cells that float in the culture container, such as dead cells. Thus, the necessary attached cells are further enriched. A person skilled in the art carries out optional step (e) at an appropriate time within the preferred range of 6 hours to 3 days, depending on the actual need to perform such a step. Whether such a possible step (e) is really necessary can be determined, for example, by observing the cells under a microscope and determining the number of non-adherent cells in the cell culture.

In another preferred embodiment of the present invention, after step (e), in some cases, an additional step (g) of expanding the adherent cells from step (e) is performed, while the propagation step (h) includes culturing the adherent cells for 1 to 5 passages up to 70- 80% confluence.

In accordance with the present invention, the person skilled in the art can determine the necessary duration and number of passages to achieve the desired 70-80% confluency.

The second object of the present invention is directed to SMDC, where SMDC contain at least 60% CD56-positive and 60% A2B5-positive cells.

In another preferred embodiment of the invention, the SMDCs are CD105 positive cells, which means that the SMDCs express CD105 on their cell surface.

In a preferred embodiment of the invention, at least 60%, 70%, 80%, 90%, 95%, or 98% of the cells, or any number of cells in between the indicated SMDC values, are CD105 positive, which means that the SMDCs express CD105 on their cell surfaces.

CD105 is also known as Endoglin. It is an integral type I membrane homodimeric protein with 90 kD subunits found on vascular endothelial cells and placental syncytiotrophoblasts. CD105 is weakly expressed on stromal fibroblasts. It is also expressed on activated monocytes and tissue macrophages. CD105 expression is upregulated on activated endothelium in tissues undergoing angiogenesis, such as tumors, or in cases 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 does not bind to TGF-β2. TGF-β receptors have been shown to be required for skeletal muscle differentiation. Indeed, the TGFβ receptor has been shown to be required to increase myogenin, a skeletal muscle differentiation factor, and the ability of myoblasts to fuse. Thus, CD105 positive cells may be preferred for SMDCs and SMDCs produced by the methods disclosed in the present invention.

In another preferred embodiment of the invention, the SMDCs are desmin positive, meaning that the SMDCs express desmin on their cell surface.

In a preferred embodiment of the invention, at least 60%, 70%, 80%, 90%, 95%, or 98% of the cells, or any number of cells in between the indicated SMDC values, are desmin-positive, which means that the SMDCs express desmin at their cell surface.

In another preferred embodiment of the invention, the SMDCs are Pax7 positive, which means that the SMDCs express Pax7 on their cell surface.

In another preferred embodiment of the invention, the SMDCs are Myf5 positive, which means that the SMDCs express Myf5 on their cell surface.

A2B5 reactive antigens belong to the c-series ganglioside family, a family of glycolipids most abundant in the nervous system, where they exhibit functions such as intercellular adhesion and recognition, as well as signal transduction. Loss of gangliosides leads to severe neurological defects such as regenerative motor neuron defects in c-series ganglioside-deficient mice (R. K. 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). Also in the neuromuscular junction (NMJ), a large number of gangliosides have been found. Treatment of NMJ with anti-ganglioside antibodies resulted in a severe defect in NMJ, thus supporting the idea that gangliosides are required for NMJ (J.J. Plomp and H.J. 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 considered to have regenerative potential (R. K. 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 sialtransferases. Among them, ST3GAL1 and ST3GAL2 have the highest in vitro glycosphingolipid acceptor activity and are highly expressed in the brain (E. R. Sturgill et al., "Biosynthesis of the major brain gangliosides GD1a and GT1b," Glycobiology, vol. 22, no. 10, pp. 1289-1301, Oct. 2012). mRNA of both ST3GAL1 and ST3GAL2 is expressed in SMDCs obtained by the present invention and is expressed in greater amounts compared to commercially available SMDCs. Taken together, SMDCs with myogenic regenerative capacity, also expressing A2B5-reactive antigens, can not only regenerate skeletal muscle, but also provide neuromuscular support, especially at the newly formed neuromuscular junction during muscle regeneration.

SMDCs that can be used to treat muscle dysfunction, in particular to treat incontinence such as urinary incontinence and/or anal incontinence, preferably show a characteristic expression profile. Preferably, more than about 60%, 70%, 80%, 90%, 95%, or 98% of said SMDCs express CD56 and A2B5. Preferably, said SMDCs do not express CD34, Sca-1 and MyoD. Thus, the term "do not express" means that preferably less than 40%, 30%, 20%, 10%, 5% or 2% of SMDCs express these markers. The SMDC expression profile as described above can be used to determine the myogenicity index of a cell culture without requiring differentiation. Thus, this SMDC expression profile can be used to determine whether skeletal muscle-derived cells can be used to treat muscle dysfunction, in particular to treat incontinence such as urinary incontinence and/or anal incontinence.

Another object of the present invention relates to SMDC obtained in accordance with the method of the present invention.

In accordance with the present invention, SMDCs obtained by the method of the present invention can be easily distinguished from cells of the prior art, taking into account the distinctive expression of the A2B5 marker and/or enzymes that synthesize the A2B5 reactive antigen. Since A2B5-reactive gangliosides are synthesized by sialyltransferases, these sialyltransferases can thus serve as an indirect marker of A2B5 expression. Among the three important sialyltransferases ST3GAL1, ST3GAL2 and ST3GAL3, the enzymes with the highest in vitro glycosphingolipid acceptor activity highly expressed in the brain are ST3GAL1 and ST3GAL2 (E.R. Sturgill et al., "Biosynthesis of the major brain gangliosides GD1a and GT1b," Glycobiology, vol. 22, no. 10, pp. 1289-1301, October 2012). Determination of the expression of ST3GAL1 and ST3GAL2 provides indirect evidence of the expression of A2B5. Therefore, the new method of the present invention allows to obtain SMDC, which differ from the SMDC obtained by methods known in this field.

In another preferred embodiment of the present invention, SMDCs and SMDCs obtained by the method of the present invention are characterized by the expression of different markers. In accordance with the present invention, SMDCs are expected to be characterized by positive expression of one, two, three, four or five markers CD56, A2B5, CD105, desmin, Myf5 and Pax7. It is further preferred that the SMDCs and SMDCs produced by the method of the present invention are characterized by the expression of a combination of different markers selected from positive expression of one or more markers selected from CD56, A2B5, CD105, Myf5 and Pax7 and negative expression of at least one a marker selected from CD34 and MyoD. Preferably, at least 60%, 70%, 80%, 90%, 95%, or 98%, or an amount in any intermediate range between the indicated SMDC values, are positive for any one or more of CD56, A2B5, CD105, Myf5, and Pax7 which means that SMDCs express CD56, A2B5, CD105, Myf5 and/or Pax7 on their cell surface. It is also preferred that no more than 59%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% or 0% of the cell population express any of CD34 and/or MyoD.

In a particularly preferred embodiment of the invention, the SMDCs and SMDCs obtained by the method of the present invention are characterized by positive expression of CD56, A2B5, CD105, Myf5, Pax7 and negative expression of CD34 and MyoD.

Another object of the present invention relates to SMDC, obtained in accordance with the method of the present invention, for use as a pharmaceutical composition.

In accordance with the present invention, SMDCs obtained in accordance with the method of the present invention can be used as an active ingredient in a pharmaceutical composition.

Another object of the present invention relates to SMDC, obtained in accordance with the method of the present invention, for use in a method for improving the neuro-muscular connection.

Preferably, the SMDCs of the present invention are for use in a method for improving the prevention and/or treatment of neuromyopathies and/or myopathies. Preferably, the SMDCs are for use in a method for the prevention and/or treatment of neuromyopathies and/or myopathies such as fecal incontinence and/or urinary incontinence.

In accordance with the present invention, SMDC can be used in a method that preferably involves the injection of SMDC to a subject suffering from incontinence. In general, injection of SMDC into a given tissue or lesion contains a therapeutically effective amount of cells in solution or suspension, preferably from about 1×10 ⁵ to about 6×10 ⁶ cells per 100 μl of injection solution. The solution for injection is a physiologically acceptable medium with or without autologous serum. A physiologically acceptable medium may be, as a non-limiting example, saline or phosphate buffer solution.

Finally, another object of the present invention relates to SMDC, obtained in accordance with the method of the present invention, for use in the treatment of muscle dysfunction, where the muscle dysfunction is incontinence, in particular urinary incontinence and/or anal incontinence.

In principle, any type of anal incontinence can be treated, as strengthening the anal muscular systems allows better control of rectal filling. However, anal incontinence resulting from perineal rupture is also treated, especially if the anal sphincter system and/or M. puborectalis is damaged and traumatized. Such a perineal tear can be caused by a wide variety of causes, as described above. However, the cause of such perineal tear is not limited to the use of the methods and SMDCs of the present invention. Patients can be treated with the methods and SMDC of the present invention if they are suffering from a third or fourth degree perineal tear. This applies in particular to women who suffer from such a perineal tear after childbirth with tweezers, give birth for the first time, give birth to a baby weighing over 4 kg, or suffer consequences due to the abnormal position of the baby before birth. The methods and SMDCs of the present invention may also be used following trauma to the anal sphincter system and/or M. puborectalis due to surgical procedures. In addition, the methods and SMDCs of the present invention can also be used in cases of temporary incontinence only. This treatment prevents the development of complete anal incontinence. In addition, anal incontinence diseases to be treated with the methods and SMDCs of the present invention are passive incontinence, fecal incontinence, and urge to defecate.

It should be understood that the methods and SMDC of the present invention can not only be used to treat patients already suffering from anal incontinence, i.e. exhibiting symptoms of anal incontinence, but can be applied to subjects not yet suffering from anal incontinence, but with an increased risk of this condition, for example, in cases where the rectal muscles have suffered from surgery, childbirth, accidents, etc. Another example would be when the rectal musculature becomes thinner than in a healthy subject, or when it undergoes degeneration for other reasons. The methods of the present invention may provide suitable prophylaxis to prevent the onset of anal incontinence.

SMDCs for use in the treatment or prevention of incontinence are preferably homologous to the recipient.

In a more preferred embodiment, said SMDCs are autologous or heterologous to the recipient. These SMDCs can be obtained, for example, by biopsy from the recipient's biceps. Autologous SMDCs reduce or minimize the risk of allergic reactions after administration of SMDCs to a recipient. Preferably, SMDCs are multinucleated fusion competent cells or myogenic cells such as myoblasts. More preferably, said SMDCs are human cells.

Therefore, the present invention also provides a simple prophylactic approach or method for treating women and men with urinary incontinence and/or anal incontinence or at risk of developing urinary incontinence and/or anal incontinence using autologous skeletal muscle-derived cells to strengthen their urinary excretion. and/or anal sphincters. This therapy, using cells derived from muscles, allows you to repair and improve the damaged urinary and anal sphincter. In accordance with the present invention, treatment includes needle aspiration to obtain muscle cells, for example, and rapid post-treatment to administer the cultured and prepared cells to the patient. Also, according to the present invention, autologous skeletal muscle-derived cells (SMDC) harvested from and cultured for a particular patient with urinary and/or anal incontinence can be used as a non-allergenic agent to increase muscle mass in the wall of the urinary system and/or rectum. intestines, thereby increasing coaptation and improving the sphincter muscles of the urinary system and/or rectum. In this aspect of the invention, a simple transplantation of autologous muscle cells is performed as described above.

In accordance with the present invention, autologous cells derived from skeletal muscle and injected directly into the urinary sphincter and/or anal sphincter demonstrate prolonged survival. Thus, injection of autologous myoblasts results in a safe and non-immunogenic long-term survival of the muscle fiber in the urinary and/or anal sphincter.

In a specific embodiment of the invention, from about 50 to about 200 µl of a suspension of cells derived from skeletal muscle (at a concentration of from about 1x10 ⁵ to about 6x10 ⁶ cells per 100 µl of injection solution) is injected into the urinary sphincter. The injection device may be connected to a container containing the cell suspension to be injected. For the treatment of anal incontinence, preferably from about 50 μl to about 1 ml, more preferably about 0.5 ml of a suspension of cells derived from skeletal muscle (at a concentration of from about 1x10 ⁵ to about 6x10 ⁶ cells per 100 μl) is injected into the external anal sphincter. The injection device may be connected to a container containing the cell suspension to be injected.

In another embodiment, the injection step may include several separate injections, for example, from about 20 to about 40 injections of a suspension of cells derived from skeletal muscle, where in each injection about 50 to about 500 μl of a suspension of cells derived from skeletal muscle are injected, and at the same time each injection is administered to a different area of the anal sphincter. However, these parameters should be considered only as an example, and the specialist will easily be able to adapt these procedures to the treatment requirements for each individual patient.

In another embodiment of the present invention, the movement of the injection device towards the urinary and/or anal sphincter is controlled by sonography and/or EMG (electromyography). In a specific embodiment, a transrectal probe is inserted and the position of the transrectal probe is optimally adjusted to treat the urinary and/or anal sphincter using the methods of the invention. In another specific embodiment, cells derived from skeletal muscle are implanted in the region surrounding the urinary and/or anal sphincter defect and/or especially in the region of the urinary and/or anal sphincter defect. The patient may start the day following the cell injection with exercise to further treat urinary and/or anal incontinence in accordance with the invention.

In another embodiment, the treatment is repeated. Treatment can be repeated, for example, within one year after the last treatment, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month(s) after the last treatment, or within 1-8 weeks, preferably 2-3 weeks or 10-20 days after the last treatment. In particular, the treatment can be repeated within 2-3 weeks after the last treatment with cells from the same cell culture that was used in the previous treatment. This approach reduces the volume of injection per injection and gives the cells more time to adapt, integrate and build muscle. In an even more specific embodiment, the injections are repeated at intervals of 2-3 weeks until an improvement in urinary and/or anal regulation is achieved.

As mentioned above, a specific route of penetration is through the skin of the patient parallel to the direction of the rectum. However, it is also contemplated that penetration may occur directly from the rectum near the injured muscle. In particular, the penetration and injection process is monitored by means of ultrasound imaging. In addition, an alternative route of penetration is being considered for women, ie. transvaginal injection. In this case, the injection device penetrates the vaginal wall and moves forward until it reaches the desired injection site. In particular, the penetration and injection process is also monitored by sonographic and/or EMG (electromyographic) imaging tools in this case.

In another embodiment, the injection includes the injection of cells derived from skeletal muscle, in the form of an "injection track". As used herein, "injection lane" refers to the arrangement of cells along the length or part of the length of the injection track, ie. along the channel formed by the introduction of a needle into the muscle tissue. In other words, after the injection, the needle is withdrawn, and at the same time, the cells are ejected from the syringe in a continuous or intermittent manner, while the injection needle is moved, in particular withdrawn along the injection track. This even distribution of the cells ensures continuous delivery of the injection solution containing the cells along the injection channel that is formed when the injection device/needle enters the target muscle tissue. In a specific embodiment, the injection path or channel should have a diameter of no more than about 0.8 mm, otherwise it may lead to necrosis of cells derived from skeletal muscle in the center of such injection channel and, therefore, lead to adverse inflammation and other processes.

The injection device for use with the methods of the present invention can be any device capable of penetrating human tissue and capable of delivering solutions, in particular solutions containing cells derived from skeletal muscle, to the desired location in the body of a subject, in particular a human. The injection device may comprise, for example, a hollow needle. The injection device may also be any type of syringe suitable for injecting cells derived from skeletal muscle. In more complex embodiments, the injection device may, for example, be a syringe gun that injects the suspension by applying air pressure. In particular, the injection device is suitable for minimally invasive applications and minimally invasive surgery, respectively.

By selecting an injection needle having a particular diameter, the injection volume per mm ³ can be accurately predetermined. The diameter of the injection needle usually does not exceed 5 mm, as this can lead to damage to muscle structures.

Ultrasound imaging to monitor the position and operation of the injection device can be achieved using any standard ultrasound imaging device known in the art. In addition to mono- or dual-plane standard ultrasound probes, new ultrasound technologies can also be used, such as for example 3D sonography or color Doppler sonography, etc. In a specific embodiment, as described above, the injection device contains means of ultrasound imaging.

It is assumed that any method or composition described here can be implemented in relation to any other method or composition described here.

The following examples illustrate the present invention, but are not intended to be limiting.

Example 1 - Receive SMDC

To obtain SMDC, a skeletal muscle biopsy was taken from M. pectoralis major or M. biceps brachii from an incontinent patient. To obtain a biopsy, the skin was first opened with an incision about 1 cm long above the muscle until the fascia of M. pectoralis major was reached. After opening the fascia, 1 cm ³ of muscle tissue was taken (biopsy). The biopsy was directly transferred to biopsy transport medium pre-cooled to about 4° C. and containing F10 Ham's basal medium supplemented with gentamicin (final concentration 1-5 μg/ml). The biopsy was stored for about 26 hours at 1-11° C. in biopsy transport medium. The biopsy was then transferred to a Petri 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 Petri dish filled with 1x PBS and dissected into 2-3 mm ² pieces using a scalpel. After an additional transfer step as described above, the tissue pieces were further cut into 1 mm pieces. These pieces were finally transferred to a centrifuge tube filled with 1x PBS and centrifuged for 10 minutes at 1300 rpm. After centrifugation, the supernatant was removed and the muscle tissue was resuspended in 1x PBS supplemented with 8 μg/ml gentamicin. Then the suspension of muscle tissue was cooled to 2-8°C for 48 hours. After cooling, the suspension of muscle tissue was centrifuged for 10 minutes at 1300 rpm, the supernatant was then removed and 2.5 ml of a digestion solution containing 1-5 mg/ml collagenase, 2-4% v/v was added. buffer Hepes, 0.1-10% vol./about. fetal bovine serum and 5-10 µg/ml gentamicin in Ham's F10 medium. The muscle cell suspension was then incubated for 6-20 hours at 37°C, 5% CO ₂ . Then the suspension was centrifuged at 1300 rpm for 10 minutes, the supernatant was removed, the pellet was resuspended in a medium containing 10-20% v/v. FCS, 1-3 ng/ml bFGF and 3-10 μg/ml gentamicin in F10 Ham's medium and plated in cell culture flasks. The SMDCs attached to the bottom of the cell culture flask were further maintained by changing media every 3-4 days and subculturing after cell separation after reaching confluency. Subculturing was performed until reaching 1×10 ⁷ - 5×10 ⁷ SMDC.

Cell counts were performed according to the Chemometec Nucleocounter™ manual. This method uses propidium iodide staining of nuclei and calculates the number of cells in 0.2 ml. Before automatic counting, 100 µl of the cell suspension was 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-Stabilizing buffer was added. The suspension was mixed, collected with a nucleocasette™ and finally measured.

On FIG. 1 shows the morphology of SMDC in standard cell culture flasks prepared by the present invention, visualized by phase contrast microscopy.

Example 2 - Flow Cytometry

Flow cytometric analysis was performed on a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, cells were harvested with trypsin at 37° C. for 5 min, centrifuged at 400 rcf (relative centrifugal force) and resuspended in 1x PBS supplemented with 1% FCS. Cells at 40,000/reaction were incubated with 5 µl of anti-CD56-PE (Beckman Coulter Inc., France), IgG1-PE isotype (Beckman Coulter), Alexa488 isotype (Sigma), anti-CD105-PE (Beckman Coulter Inc., France). , France) or A2B5-A1exa488 antibody (Millipore) for 15 minutes in a 1.5 ml Eppendorf® tube at 4°C in the dark. Cells were washed with 1 ml PBS, centrifuged at 400 rcf and resuspended in 200 μl 1x PBS for FACS analysis in a 96 well round bottom plate. After washing and resuspension, 5 μl of 7-amino-actinomycin D dye (Beckman Coulter Inc., France) was added to each reaction mixture to determine cell viability and the plate was incubated for 10 minutes at 4°C. Cellular events were determined using Guava InCyte™ v.2.3 software. Histograms and scatter plots were generated with a minimum number of 3000 events at a sample flow rate of 1.8 μl/ml. Positive staining was obtained by comparing the isotype control, established as at least 99% negative.

Example 3 - Immunocytochemistry

First, the supernatant was removed from the cell culture dishes and the cells were washed three times with PBS. Permeabilization and fixation was performed by coating the cells with 4% formaldehyde solution (v/v; diluted in PBS) for 20 minutes at room temperature. The cells were then washed with PBS three times after each incubation step. After that, the cells were covered with 500 μl of hydrogenperoxid-block (Thermo Fisher Scientific) (blocking solution based on hydrogen peroxide) and incubated for five minutes at room temperature. Cells were pipetted with primary antibodies (desmin) at a final concentration of 40 μg/ml (w/v) and incubated for at least 90 minutes (37° C., 5% CO ₂ ). The cells were then coated with 500 μl of biotin-conjugated secondary antibodies (goat vs. rabbit, polyclonal, Thermo Fisher Scientific) and incubated under the same conditions as for the primary antibodies, but for at least 60 minutes. To visualize antibody binding, 500 μl Streptavidin Horseradish Peroxidase Conjugate (Vectorlabs) at a final concentration of 2-5 μg/ml (diluted in PBS) was added and incubated at 37°C, 5% CO ₂ for 20 minutes followed by by applying to the cells 500 μl of a one-component solution with a chromogen, which was removed after 5-15 minutes. A final washing step with PBS was performed before results could be observed. Cells stained as desmin positive by immunocytochemistry were dark red.

Example 4 Purity of SMDC

In SMDC obtained by the method described in Example 1, their purity was determined by myogenic markers CD56 (NCAM) and desmin. The percentage of CD56 positive cells was determined by flow cytometry as described in Example 2. SMDCs prepared according to the present invention as described in Example 1 contain 99.3% CD56 positive cells (Fig. 2A) and 98.77% viable cells ( Fig. 2B). In addition, desmin expression in one representative batch of SMDC obtained by the method described in Example 1 was analyzed by immunocytochemistry as described in Example 3. FIG. 4 shows the high purity of the SMDCs produced in Example 1 as most of the cells are positive for desmin staining (FIG. 3).

Example 5 Cell Differentiation

Differentiation of human myoblasts into syncytial myotubules occurs when the growth medium is replaced with serum-free differentiation medium. To Skeletal Muscle Cell Differentiation Medium (Promo Cell) was added Skeletal Muscle Cell Differentiation Medium Supplement Pack (as described in the protocol of the company from which the medium was purchased) and 250 μl of gentamicin. To start differentiation, cells (in growth medium) were seeded in 4-well or 24-well plates (60,000-480,000 cells) pre-counted as described in Example 1. After cells were attached to the well (overnight), the growth medium was discarded , cells were washed once with differentiation medium and covered with differentiation medium with a volume of 500 μl.

Example 6 - Myogenic activity of SMDC

SMDC cells, which belong to the myogenic lineage, are able to fuse and form syncytial myotubules in vitro, which are characteristic of skeletal muscle fibers in vivo. Thus, the myogenic activity of SMDC was tested using in vitro differentiation. In the resulting cells described in Example 1, differentiation was induced as described in Example 5 to determine myogenic activity. The SMDCs produced huge myotubules that were desmin positive (stained as described in Example 3) as seen from the representative batch of SMDCs in FIG. 4. The results confirmed the myogenic activity of SMDC, which is essential for functional regeneration of skeletal muscle tissue.

Example 7 Gene Expression Analysis

The SMDC cells prepared in Example 1 were seeded, followed by cell counts also described in Example 1, at a density of 500,000 cells/well in 6-well plates (NUNC Thermo Scientific) to 70% confluence, then total RNA was isolated using an isolation kit RNeasy RNA (QIAGEN) according to manufacturer's instructions. RNA concentration and purity were assessed by microfluidic analysis using denaturing gel electrophoresis and an Agilent 2100 bioanalyzer. Sample preparation for microarray hybridization was performed as described in the NuGEN Ovation PicoSL WTA System V2 and NUGEN Encore Biotin Module manuals (NuGEN Technologies, Inc, San Carlos, CA, USA). Briefly, 7.5 ng of total RNA was reverse transcribed into double stranded cDNA in a two step process introducing the SPIA tag sequence. The bead-purified cDNA was amplified by the SPIA amplification reaction followed by further purification with microspheres. 4.5 μg SPIA cDNA was fragmented, terminally labeled with biotin and hybridized with Affymetrix Prime View Human Gene Expression arrays for 16 h at 45°C in a GeneChip Hybridization Oven 640 hybridization chamber. Hybridized panels were washed and stained with Affymetrix Fluidics Station FS450, and fluorescent signals were measured using Affymetrix GeneChip Scanner 3000 7G. The inkjet and scanning functions were controlled using the 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.de). The total signals of the set of probes in the log2 scale were calculated using the RMA algorithm (1) using the Affymetrix GeneChip Expression Console v1.4 software. To compare the expression results of the SMDC genes obtained in Example 1 with the SMDC genes obtained by methods known in the art, the gene expression data of commercially available SMDC (Lonza) published in Abujarour et al., 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 07/21/2017). Comparison of the expression of the SMDC genes obtained in Example 1 with the SMDC genes analyzed by Abujarour et al., 2014 was performed by comparing mean reads per annotation of different genes using Partek Flow software according to the company that provided it (Partek Inc.). Higher mean readings per annotation thus represent a higher amount of annotated mRNA corresponding to higher gene expression.

Example 8 Characterization of SMDC Neuromuscular Support

The neuromuscular support characterization of the SMDCs obtained using Example 1 was determined by the positive binding of the A2B5 antibody to the SMDC cell surface by flow cytometry (described in Example 2). As shown in FIG. 5, 96.16% of the SMDCs obtained in Example 1 are positive for A2B5 reactive antibody, demonstrating that in addition to their myogenic activity (expression of desmin, CD56), SMDCs also exhibit neuronal characteristics. To compare the neuromuscular support characteristics of SMDCs prepared according to the present invention with myoblasts isolated by methods known in the art, the gene expression of enzymes necessary for the formation of A2B5-reactive gangliosides, exhibiting the highest in vitro glycosphingolipid acceptor activity ST3GAL1 and ST3GAL2 SMDCs, analyzed as described in Example 7. mRNA expression of both ST3GAL1 and ST3GAL2 was higher in SMDCs prepared according to the present invention compared to commercially available SMDCs, as shown in FIG. 5. In addition, ST3GAL3 expression was also higher in the SMDCs obtained in Example 1.

Example 9 - Obtaining cells without cooling after tissue dissection

As a comparative example, the method of the present invention was carried out with the omission of the cooling step. To obtain SMDC, a skeletal muscle biopsy was taken from M. pectoralis major or M. biceps brachii of an incontinent patient. To obtain a biopsy, the skin was first opened with an incision about 1 cm long over the muscle until the fascia of M. pectoralis major was reached. After opening the fascia, 1 cm ³ of muscle tissue was taken (biopsy). The biopsy was directly transferred to biopsy transport medium pre-chilled to about 4° C. and containing F10 Ham's basal medium supplemented with gentamicin (final concentration 1-5 μg/ml). The biopsy was stored for about 26 hours at 1-11° C. in biopsy transport medium. The biopsy was then transferred to a Petri 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 Petri dish filled with 1x PBS and dissected into 2-3 mm ² pieces using a scalpel. After an additional transfer step as described above, the tissue pieces were further cut into 1 mm ² pieces. These pieces were finally transferred to a centrifuge tube filled with 1x PBS and centrifuged for 10 minutes at 1300 rpm. After centrifugation, the supernatant was removed and the muscle tissue was resuspended in 1x PBS supplemented with 8 μg/ml gentamicin. The suspension of muscle tissue was centrifuged for 10 minutes at 1300 rpm, then the supernatant was removed and 2.5 ml of a digestion solution containing 1-5 mg/ml collagenase, 2-4% v/v was added. buffer Hepes, 0.1-10% vol./about. fetal bovine serum and 5-10 µg/ml gentamicin in Ham's F10 medium. Then the suspension of muscle tissue was incubated for 6 to 20 hours at 37°C, 5% CO ₂ . Then the suspension was centrifuged at 1300 rpm for 10 minutes, the supernatant was removed, the pellet was resuspended in a medium containing 10-20% v/v. FCS, 1-3 ng/ml bFGF and 3-10 μg/ml gentamicin in F10 Ham's medium, and placed in cell culture flasks. SMDCs attached to the bottom of the culture flask were maintained with medium change every 3-4 days and subcultured followed by separation after reaching confluence. Subculturing was performed until reaching 1×10 ⁷ - 5×10 ⁷ SMDC cells.

Example 10 - Expression of the Mesenchymal Cellular Marker SMDC

Expression of the mesenchymal marker of SMDCs obtained using Example 1 was determined by binding an anti-CD105 antibody, anti-CD105-PE (Beckman Coulter Inc., France), to the cell surface of SMDCs measured by flow cytometry (described in Example 2). As shown in FIG. 8, 98.69% of the SMDCs obtained in Example 1 were determined to be positive for CD105-reactive antibody when the positivity threshold was set by isotope control (0.31% positive), indicating that the SMDCs are mesenchymal cells and/ or are of mesenchymal origin.

Example 11 Measurement of Acetylcholinesterase Activity of SMDC

To measure acetylcholinesterase activity (AChE), 200,000 SMDC cells were seeded in a gelatin-coated well of a 24-well plate and differentiation was induced as described in Example 5. Six days after the start of differentiation, the differentiation medium was carefully removed from the 24-well plate with an immediate addition of 300 μl 0.5 mM solution of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) (prepared in phosphate buffer, pH 7.2, with 0.1% Triton X-100). After 2 minutes of incubation at room temperature in the dark, 50 μl of 5.76 mM acetylthiocholine iodide (ATI) (prepared in distilled water) was added. The contents of the reaction mixture were incubated for 60 minutes at 30°C in the dark, followed by measurement of optical density (OD) at 412 nm (OD412 nm) on an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK). A control reaction was also included, which consisted of all reagents except cells. Corrected OD412 nm values were obtained by subtracting the mean control measurement from the mean OD412 nm value. AChE activity in relative mU (AChE mUrel) was calculated according to the straight line equation for the standard curve. A standard curve was generated by measuring 4-500 mU/ml dilutions of ready-to-use stock solution of 50 U/ml AChE (from electric eel, AAT Bioquest® Inc., Sunnyvale, CA, USA). 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 OD412 nm was measured after 60 minutes in a 24-well plate. To normalize for protein concentrations, for cell type, the AChE activity of the measured number of cells was divided by the total protein content of those cells, resulting in AChE mUrel activity per g protein (AChE mUrel/g protein). To analyze the total protein in the cell population, an aliquot of adherent cells differentiated according to Example 5 was first washed twice with PBS, then coated with PBST (0.1% X-100 Triton) and then incubated for 10 minutes at room temperature. The lysate was then resuspended and transferred to an Eppendorf® tube, shaken briefly and then centrifuged for 4 minutes at 1200*g. Finally, the clear supernatant was transferred to a fresh Eppendorf® tube and the protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific, MA, USA) according to the manufacturer's instructions by measuring the OD at 540 nm using an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK).

Example 12 - Quantifying SMDC Merger Competence

The fusion competence of SMDCs is based on their ability to form multinucleated myotubules and represents their myogenic activity. Fusion competence is defined as the fusion index, which is the number of nuclei located in multinucleated myotubules, defined as cells containing at least 3 nuclei divided by the total number of nuclei in the visible field of the microscope. To determine the SMDC confluence index, 2*105 cells were plated in gelatin-coated wells of 24-well plates and differentiation was induced by replacing the growth medium with skeletal muscle differentiation medium 24 hours after seeding. After 6 days of differentiation, cells were washed twice with PBS and fixed with 4% PFA for 10 minutes. Cells were then washed three times with PBS and stained with 2 μg/ml Hoechst33342 solution for 20 minutes. For each sample, at least three fields were fixed during immunofluorescence imaging and superimposed on phase contrast images, which made it easy to detect nuclei and cell boundaries. The fusion index was calculated for each fixed field of view by dividing the number of nuclei within the tubules by the total number of nuclei per field, followed by the calculation of the average for all analyzed fields. Only cells with at least 3 nuclei were considered as myotubules. For statistical analysis, at least 3 populations from different patients were analyzed for each cell group.

Claims (22)

1. Method for obtaining a population of skeletal muscle derived cells (SMDCs) having myogenic activity and fusion competence, wherein the population contains at least 90% CD56 positive, 90% A2B5 positive, 90% CD105 positive, and 90% desmin -positive cells, including the stages: (a) cooling the sample obtained from skeletal muscle tissue in buffer; (b) processing with subsequent cooling of the sample; where the cooling is performed at a temperature of from 1 to 16°C for a period of time from 2 hours to 48 hours; (c) resuspending the sample from step (b) in serum containing collagenase medium and heating to 25-38°C for 1-20 hours; sedimentation of the sample by centrifugation; And (d) resuspending the pellet of the sample from step (c) to obtain a single cell suspension from the sample from step (c), thereby obtaining a population of SMDCs. 2. The method according to p. 1, where stage (a) is performed at a temperature below 16°C, preferably in the temperature range from 1 to 16°C, preferably from 4 to 10°C, particularly preferably at 7°C; and for a period of time up to 96 hours. 3. The method according to claim 1 or 2, wherein step (b) comprises the use of scissors, a scalpel, tweezers, a filter or a ball mill. 4. The method according to any one of paragraphs. 1-3, where the cooling in step (b) is carried out at a temperature in the range from 4 to 8°C, particularly preferably at 4°C and for a period of time from 2 hours to 48 hours. 5. The method according to any one of paragraphs. 1-4, where stage (c) includes carrying out an enzymatic treatment with a solution containing one or more enzymes selected from the group consisting of trypsin, papain, elastase, hyaluronidase, deoxyribonuclease and DNase. 6. The method according to any one of paragraphs. 1-5, where stage (c) is carried out at a temperature in the range from 36 to 38°C, particularly preferably at 37°C. 7. The method according to any one of paragraphs. 1-6, wherein step (d) preferably comprises a method selected from at least one of FACS sorting (fluorescence activated cell sorting), centrifugation, electrokinetic sorting, acousticophoresis sorting, microsphere cell sorting, and optical sorting. 8. The method according to any one of paragraphs. 1-7, where after step (d) an additional step (e) is performed, including incubation of the single cell suspension obtained in step (d), where the incubation in step (e) is preferably performed at a temperature in the range from 25 to 38°C, preferably from 36 to 38°C, particularly preferably at 37°C, thus obtaining attached SMDCs. 9. The method according to claim 8, where after step (e) an additional step (e) is performed, including the removal of cells not attached in step (e), where step (e) is preferably performed after a period of time from at least 6 hours to 4 days. 10. The method according to claim 9, where after stage (e) an additional stage (g) of propagation of cells attached in stage (e) is performed, while propagation in stage (h) includes cultivation of attached cells for 1-5 passages up to 70 -80% confluence. 11. Cell population of SMDCs (cells derived from skeletal muscle) with myogenic activity and fusion competence, where the population contains at least 90% CD56-positive, 90% A2B5-positive, 90% CD105-positive and 90% desmin- positive cells. 12. A cell population of SMDCs according to claim 11, wherein more than 95% or 98% of said SMDCs express CD56, A2B5, CD105 and desmin. 13. The cell population of SMDCs according to claim 11 or 12, obtained by the method according to any one of paragraphs. 1-10. 14. Cell population SMDCs according to any one of paragraphs. 11-13 for use as a pharmaceutical composition. 15. Cell population SMDCs according to any one of paragraphs. 11-13 for use in a method for preventing or treating neuromyopathies and/or myopathies; preferably for use in a method for the prevention or treatment of neuromyopathies and/or myopathies, in particular fecal incontinence and/or urinary incontinence. 16. Cell population SMDCs according to any one of paragraphs. 11-13 for use in a method of treating muscle dysfunction, wherein the muscle dysfunction is incontinence, in particular urinary incontinence and/or anal or fecal incontinence. 17. The cell population of SMDCs for use according to claim 15 or 16, where the cell population according to any one of paragraphs. 11-13 is intended for use in a method for treating urinary incontinence and the cell population of SMDCs is provided in 50-200 µl of a cell suspension injection solution at a concentration of 1x105 to 6x106 cells per 100 µl of an injection solution for injection into the urinary sphincter. 18. The cell population of SMDCs for use according to claim 15 or 16, where the cell population according to any one of paragraphs. 11-13 is intended for use in a method for the treatment of anal incontinence and the cell population of SMDCs is provided in 50 µl - 1 ml of cell suspension injection solution with a concentration of 1x105 to 6x106 cells per 100 µl injection solution for injection into the external anal sphincter, and in particular, wherein the method for treating anal incontinence comprises 20 to 40 injections of a skeletal muscle cell suspension, wherein 50 to 500 µl of a skeletal muscle cell suspension is injected in each injection, and where each injection is administered to a different region of the anal sphincter.

Images