Method for obtaining induced smooth muscle cells

Abstract

The present invention relates to methods for obtaining induced smooth muscle cells (iSMCs), iSMCs, iSMCs for use in methods of treating a disease or disorder or in tissue engineering, and the use of skeletal muscle-derived cells in obtaining iSMCs.

Description

[0001] The present invention relates to a method for obtaining induced smooth muscle cells (iSMCs), iSMCs, iSMCs for use in a method of treating a disease or disorder or for use in tissue engineering, and the use of skeletal muscle-derived cells in obtaining iSMCs.

[0002] Degeneration of smooth muscle, such as in sphincters, can lead to debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells (SMDCs) have been effectively used clinically to regenerate skeletal muscle sphincters, such as the external anal or urethral sphincter. However, little is known about the in vitro smooth muscle differentiation and in vivo smooth muscle regeneration potential of SMDC-derived smooth muscle cells.

[0003] Sphincters are circular muscles that control the movement of solids and / or liquids and can be composed of skeletal muscle (e.g., the external anal sphincter) or smooth muscle (e.g., the internal anal sphincter and the pyloric sphincter) (Al-Ali et al., 2009; Ramkumar & Schulze, 2005). Dysfunction of the anal and pyloric sphincters is associated with fecal incontinence and gastroparesis, respectively (Abrahamsson, 2007; Rao, 2004). Degeneration of the smooth muscle of the internal anal sphincter is a known cause of the major type of fecal incontinence - passive fecal incontinence, affecting 78% of all patients with fecal incontinence (Mimura et al., 2004). Although not life-threatening, fecal incontinence severely affects the quality of life of patients (Meyer & Richter, 2015) and has a prevalence of up to 12% in both men and women (Goode et al., 2005; Quander et al., 2005). Conservative therapies such as the application of fillers have had limited success in patients with high severity of incontinence, and surgical methods have a high morbidity and complication rate (J.Y. Wang & Abbas, 2013).

[0004] The functionality of smooth muscle tissue depends on the presence of highly differentiated smooth muscle cells that express contractile proteins, such as smooth muscle actin alpha (aSMA), desmin, and smoothelin (SMTN) (Capetanaki et al., 1997; van Eys et al., 2007; J. Wang et al., 2006), as well as functional voltage-gated calcium and potassium channels, enabling the induction of regulated cell contraction (Sanders, 2008). The isolation and application of similar cells for the treatment of smooth muscle defects may be a promising therapeutic option. However, there is currently no smooth muscle cell therapy on the market. Isolating smooth muscle cells that can regenerate defective smooth muscle tissue is a primary prerequisite for the clinical application of these cells. The existing techniques for deriving smooth muscle cells use smooth muscle tissue as a source. These primary smooth muscle cells have been effectively used for smooth muscle regeneration in animal models of passive fecal incontinence (Bohl et al., 2017), but primary smooth muscle cells are difficult to access in living humans for autologous therapy, are inherently heterogeneous, and may be limited in their proliferative capacity (Sandison & McCarron, 2015), and thus are less suitable as cell therapy candidates for human smooth muscle regeneration. Therefore, attempts have been made to utilize highly proliferative stem / progenitor cells that are primed to differentiate into smooth muscle cells.

[0005] Stem / progenitor cells such as multipotent mesenchymal stromal cells (MSCs) and induced pluripotent stem cells (iPSCs) have been shown to have the potential for transdifferentiation into the smooth muscle lineage (Bajpai et al., 2012; Park et al., 2013) as well as the potential for smooth muscle regeneration in vivo (Li et al., 2016). iPSCs are particularly promising in terms of their smooth muscle differentiation potential and function in vitro (Bajpai et al., 2012), and iPSC-derived smooth muscle progenitor cells have shown the potential for urethral sphincter regeneration in vivo (Li et al., 2016), but concerns about safety, such as genetic instability and teratoma formation, limit their application (Jung et al., 2012). In most clinical trials, adult MSC-derived cell products do not cause serious health problems (Y. Wang et al., 2012). However, their clinical efficacy in smooth muscle regeneration remains elusive.

[0006] Skeletal muscle tissue has been found to be a source of stem and progenitor cells, such as MSC and satellite cell-derived myogenic progenitors, both of which are expected to be highly regenerative (Yin et al., 2013). Skeletal muscle-derived cells (SMDCs) enriched in CD56-positive cells have been shown to improve fecal incontinence associated with external anal sphincter weakness in clinical practice (A. Frudinger et al., 2010, 2015; Andrea Frudinger et al., 2018). In addition, skeletal muscle-derived cells have been found to implant into the detrusor muscle of the bladder, thereby improving bladder function (Huard et al., 2002). However, current understanding of the differentiation of SMDCs into smooth muscle cells and their isolation or their in vivo regenerative capacity is limited (Lu et al., 2011), and no study has evaluated the therapeutic potential of SMDC-derived smooth muscle cells (induced smooth muscle cells) for sphincter smooth muscle regeneration.

[0007] Cell therapy methods for smooth muscle tissue regeneration are highly desirable and rely on the application of cells with the ability to regenerate smooth muscle. Given the drawbacks of existing technical methods, there is a need to provide new methods for smooth muscle cells.

[0008] Frudinger et al. (2018) taught the isolation of CD56+ skeletal muscle-derived cells, designated as SMDCs, as shown in Figure 6 (Andrea Frudinger et al., 2018). The cells are particularly characterized by negative expression of aSMA, CD49a, and CD146, as shown in FIG. 17 of the present disclosure. In addition, as described by Frudinger et al. (2018), SMDCs are characterized by positive expression of Pax-7 (Andrea Frudinger et al., 2018). The SMDCs described by Frudinger et al. (2018) are skeletal muscle-derived, i.e., they are capable of fusing to form multinucleated myotubes.

[0009] EP2206774A1 relates to a cell population with differentiation ability, which can be obtained by isolation from muscle tissue, more specifically from skeletal and / or cardiac muscle tissue, preferably from the endomysium and / or cardiac tissue (Marolleau et al., 2010). The cell population of EP2206774A1 contains ALDH-positive cells and particularly has myogenic and / or adipogenic and / or osteogenic differentiation ability. In particular, EP2206774A1 discloses ALDH+ / CD34− cells, ALDH+ / CD34+ cells, and SMALD / 34+ cells, all of which are CD146−, as shown in Tables 1 and 3 of EP2206774A1 and Figure 8As shown. In addition, EP2206774A1 discloses SMALD / 34-cells. The cells are CD146+ and have fusion ability, as shown in Figure 3 of EP2206774A1.

[0010] Lecourt S et al. (2010) studied human skeletal muscle as an important source of various cell progenitors with potential therapeutic promise. On the one hand, CD56+ cells were described, which are CD49a- and CD49e+. On the other hand, CD56- cells were described, which are CD146- and SMA- (Lecourt et al., 2010).

[0011] Thurner et al. (2018) disclosed the development of an in vitro potency assay for human skeletal muscle-derived cells. In particular, the isolation of CD56+ and CD56- skeletal muscle-derived cells (SMDC) was described. As shown in Figure 17 of the present disclosure, both the CD56+ and CD56- SMDC described by Thurner et al. (2018) are aSMA-, CD146- and CD49a-. In addition, as shown in Figure 2 a of Thurner et al. (2018), the AChE activity of CD56+ cells > 1000 mUrel / g.

[0012] The present invention forms the basis of the technical problem of providing a method for obtaining iSMC, which is safely and effectively applied in a method for treating a disease or disorder of a subject. Another potential technical problem of the present invention is to provide cells that are safely and effectively applied in the regeneration of smooth muscle tissue.

[0013] This technical problem is solved by the subject matter defined in the claims.

[0014] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

[0015] Figure 1 Characterization of skeletal muscle-derived MPCs and MSCs according to their differentiation potential.The differentiation potential of MPCs and MSCs was evaluated by culturing them in their respective differentiation media and differentiating them into adipogenic, chondrogenic, osteogenic, and skeletal muscle-derived lineages in vitro, and was detected by Oil Red O (adipocytes), Alcian Blue (chondrocytes), Alizarin Red S (osteocytes), and anti-desmin / Hoechst (nuclei and myocytes) staining, respectively. Representative images of at least three individually prepared samples are shown (scale bar = 100 μm) (A). The adipogenic, chondrogenic, osteogenic, and skeletal muscle-derived differentiation potential of MSCs and MPCs was quantified by calculating the average staining intensity of Oil Red O, Alcian Blue, or Alizarin Red S staining per field of view or by calculating the fusion index of at least three individual samples, respectively (B). Data are represented as the mean ± SD of MPCs and MSCs from at least three individual muscle biopsies. Statistical comparisons were performed by unpaired t-test (p < 0.05 was considered significant).

[0016] Figure 2 Display the table of skeletal muscle-derived MPCs and MSCs and their derived iSMCs by their cell surface marker expression Feature. The surface expression of mesenchymal (CD105, 90, 73), myogenic (CD56), hematopoietic (CD34), and smooth muscle lineage markers (CD146 and CD49a) on skeletal muscle-derived MPCs and MSCs and their derived iSMCs, each from at least three individual human skeletal muscle biopsies, was evaluated by flow cytometry. As a control, the expression of smooth muscle lineage markers CD146 and CD49a was confirmed in a population of human bladder-derived smooth muscle cells (hBd-SMCs). Data are represented as the mean ± SEM.

[0017] Figure 3 Show the expression of intracellular markers in SMDC (MPC and MSC) and iSMC. Detection of general myogenic (desmin) and smooth muscle myogenic (αSMA, smooth muscle actin) markers in MPCs, MSCs, and their derived iSMCs was performed by immunocytochemistry, showing representative images (scale bar = 100 μm) (A). The percentage of cells positive for αSMA, smooth muscle actin, or desmin within MPCs, MSCs, and their derived iSMCs was evaluated by quantifying the expression of the corresponding marker cells on immunocytochemistry images of cultures from at least three individual human muscle biopsies (B). Data are represented as the mean ± SEM.

[0018] Figure 4 Show the changes in gene expression during the transdifferentiation of SMDC (MPC and MSC) into iSMC.Gene expression changes were evaluated by microarray analysis of MSC and MPC cultured for 6 days in growth medium (MSC and MPC) or smooth muscle differentiation medium (MSC-iSMC and MPC-iSMC) derived from two separate human muscle biopsies to MSC-iSMC and MPC-iSMC. Gene clusters similarly upregulated (A) or downregulated (B) in both MSC and MPC after iSMC differentiation obtained by k-means clustering are depicted in the heatmap. Asterisks (*) mark genes upregulated (log2 FC ≥ 1) or downregulated (log2 FC ≤ -1) in both cell types. Statistical comparison was performed by chi-square test, and a p-value below 0.05 was considered significant. Results of gene expression changes are shown in (C).

[0019] Figure 5 Show the fusion ability of MPC and iSMC. Myotube formation (fusion ability) was observed by fluorescence microscopy after Hoechst33342 staining to visualize cell nuclei. Based on the images, the fusion index (FI) (A) and the number of nuclei per tube (B) were determined. Cells with at least 3 nuclei were counted as tubes. Measurements were compared between MPC and its derived iSMC.

[0020] Figure 6 Show the formation of functional ion channels during the transdifferentiation of MPCs into iSMCs. Voltage-dependent inward calcium (A) and outward potassium currents (B) were analyzed in MPC and its derived iSMC as well as bladder-derived smooth muscle cells (hBd-SMC). Impedance-voltage (IV) curves of at least three individual cells each of MPC, iSMC (derived from MPC), and hBd-SMC indicated the presence of functional Ca v (A) and K v (B) channels in iSMC (derived from MPC) and hBd-SMC, but not in MPC.

[0021] Figure 7 Show the contractility of SMDC and iSMC in collagen gel lattice. Contractility of SMDC (MSC and MPC), their derived iSMC, and bladder-derived smooth muscle cells (hBd-SMC) was quantified by collagen gel lattice contraction. The percentage of gel contraction from the original size within 48 hours of cells obtained by step (a) of the present invention (MPC or MSC) and cells transdifferentiated into iSMC by step (b) of the present invention as well as control smooth muscle cells (hBd-SMC) from human bladder are shown as a bar graph (A). Data are represented as the mean ± SEM of cell preparations from at least three separate human muscle biopsies or hBd-SMC analyses. Representative stereomicroscopic images of collagen gels embedded in the wells of a 24-well plate with MSC and MSC and their respective derived iSMC and hBd-SMC (B).

[0022] Figure 8 Demonstrate the smooth muscle cell phenotype of mMPC-derived iSMCs and their transplantation into smooth muscle tissue in vivo.The percentages of desmin- and α-SMA-positive cells in iSMCs derived from murine MPCs are depicted as a bar graph (A). Fluorescent signal detection of fluorescent beads and localization of TdTomato transgenic expression in the intact pyloric sphincter by in vivo imaging (B). At 12 weeks after transplantation, α-smooth muscle actin (α-SMA) protein expression, TdTomato expression, and overlay (MERGE) of TdTomato and α-SMA proteins of transplanted iSMCs were detected in tissue sections of the pyloric sphincter by immunohistochemistry. Nuclear counterstaining in each image was performed with DAPI. Representative images of n = 8 injected mice are shown (C).

[0023] Figure 9 Optical and scanning electron microscope images showing tissue rings. Optical microscopy images at different magnifications of tissue rings obtained by 3D culturing MPC-derived iSMCs around the central column of an agarose template (A and B). Scanning electron microscopy images at different magnifications of tissue rings obtained by 3D culturing MPC-derived iSMCs (C and D).

[0024] Figure 10 Show the expression of contractile proteins in SMDC- and iSMC-derived tissue rings obtained by immunofluorescence. Immunofluorescent staining of general myogenic (desmin) and smooth muscle myogenic (α-SMA) markers and nuclear counterstaining (Hoechst) were performed on cryosections of tissue rings obtained from 3D cultures of MPCs and their derived iSMCs.

[0025] Figure 11 Transmission electron microscope image showing tissue rings.

[0026] Images obtained by transmission electron microscopy of tissue rings obtained by 3D culturing iSMCs by ultrathin sectioning, showing caveolae (arrows) on the cell membranes of two adjacent cells (A and B), abundant filamentous structures in the cytoplasm (C), and densely aggregated filaments (arrows) (D).

[0027] Figure 12 List the quantification of gene expression changes during the transdifferentiation of SMDCs (MPCs and MSCs) into iSMCs. Gene expression changes were evaluated by microarray analysis of MSCs and MPCs derived from two separate human muscle biopsies cultured for 6 days in growth medium (MSC and MPC) or smooth muscle differentiation medium (MSC-iSMC and MPC-iSMC) to MSC-iSMC and MPC-iSMC. Log2 fold changes of smooth muscle-related genes in MSC compared to MSC-iSMC (MSC) and MPC compared to MPC-iSMC (MPC) samples were shown to compare smooth muscle differentiation between MSCs and MPCs. Asterisks (*) mark genes that were upregulated (Log2 ≥ 1) or downregulated (Log2 ≤ -1).

[0028] Figure 13 Showing mouse MPC-iSMCs stained with the anti-CD49e antibody of Example 15 and isotype control staining Flow cytometry analysis.

[0029] Scatter plots of IgG1 isotype control (A) and anti-CD49e (B) positive MPC-iSMCs showing 0.14% control and 9.5% CD49e positive cells, respectively.

[0030] Figure 14 Show the AChE and CK activity analysis of MPC and MPC-iSMC, in which in skeletal muscle differentiation medium (SKDiff) Measure enzyme activity before and after culturing cells.

[0031] AChE activity (A) and CK activity (B) were measured and compared in MPCs and MPC-iSMCs before and 6 days after culturing in skeletal muscle differentiation medium (SKDiff), respectively. Data are represented as mean ± SEM of cells derived from at least three separate human muscle biopsies. Statistical analysis was performed by paired t-test, considering p < 0.05 as significant.

[0032] Figure 15 Provide an overview of the characteristics and marker expression of the different cell types described herein. As described in Example 18, CD56+ SMDCs and CD56− SMDCs (Thurner et al. 2018) and SMDCs (Frudinger et al. 2018) were obtained. As described in Example 1, MPCs and MSCs were obtained. As described in Example 2, MPC-iSMCs and MSC-iSMCs were obtained. If at least 50% of the cells in the test cell population express a certain cell marker, the expression of the corresponding marker is denoted as “+”. If less than 50% of the cells in the test cell population express a certain cell marker, the expression of the corresponding marker is denoted as “−”. If, as measured according to Example 17, a certain cell population has an AChE activity of at least 1000 mUrel / mg protein, the AChE enzyme activity of that cell population is denoted as “+”. If, as measured according to Example 17, a certain cell population has an AChE activity of less than 1000 mUrel / mg protein, the AChE enzyme activity of that cell population is denoted as “−”. If, as measured according to Example 17, a certain cell population has a CK activity of at least 100 mUrel / mg protein, that cell population is denoted as “+”. If, as measured according to Example 17, a certain cell population has a CK activity of less than 100 mUrel / mg protein, the CK enzyme activity of that cell population is denoted as “−”.

[0033] When used in the claims and / or the specification in conjunction with the term “comprising”, the use of the word “a” or “an” may mean “one”, but it is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”.

[0034] The term “about” means the stated value plus or minus 5% of that value, or the standard error of measurement of a given value.

[0035] As used herein, the term "anal incontinence" refers to any unwanted discharge of bowel contents through the anus, such as gas, liquid, or solid feces. The term includes all three severity grades: grade 1 = gas only, grade 2 = liquid and soft feces, grade 3 = solid, formed feces.

[0036] As used herein, the term "anal sphincter" or "anal sphincter apparatus" specifically refers to the external anal sphincter and the puborectalis muscle as part of the levator ani muscle. However, it also includes: the pubococcygeus muscle, the ischiococcygeus muscle (M. ischiococcygeus), the iliococcygeus muscle (M. iliococcygeus), and the pudendal nerve (N. pudendus).

[0037] The term "skeletal muscle-derived cell" or "SMDC" refers to cells obtained from skeletal muscle tissue, which include fusion-capable cells such as myoblasts, or non-fusion-capable cells such as multipotent mesenchymal stromal cells, and can be primary cells and / or cells cultured in vitro or alternatively other cells with myogenic or multi-differentiation potential (e.g., from liposuction tissue or other tissues containing stem cells such as bone marrow). The term also includes fat-derived cells that can be isolated and used to differentiate into smooth muscle cells. The term "skeletal muscle-derived cell" or "SMDC" also refers to a cell population isolated from muscle tissue.

[0038] The term "human bladder-derived smooth muscle cells (hBd-SMC)" refers to a cell population containing smooth muscle cells from the human bladder. hBd-SMC can be purchased commercially (Catalog No.: C-12571) and represents the phenotypic and functional characteristics of smooth muscle cells obtained by iSMC derived from skeletal muscle-derived cells as envisaged in the present invention.

[0039] As used herein, the term "injection" refers to the discharge of an injection solution containing the above cells from an injection device into a specific site in the human body, particularly into or near muscle tissue, so as to provide anal continence. The injection process can be but is not limited to static, i.e., the injection device remains at the position it reaches. 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.

[0040] As used herein, the term "injection site" refers to the site in the human body where the injection process begins, such as near or being the muscle tissue that provides anal continence. The injection site does not need to be the same as the site where the injection process ends.

[0041] As used herein, the term "injection device" refers to any device suitable for penetrating human tissue to reach the injection site of interest and capable of delivering a solution, particularly a solution containing muscle-derived cells, to the injection site of interest.

[0042] As used herein, the term "fecal incontinence" refers only to the unwanted loss of liquid or formed feces through the anus.

[0043] As used herein, the term "passive incontinence" refers to the lack of sensory recognition of fecal loss. This includes low anal baseline pressure values due to smooth muscle defects of the internal anal sphincter and / or lack of sensory ability of the anal and rectal mucosa.

[0044] As used herein, the term "CD56+" or "CD56 positive" refers to cells that express 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.

[0045] 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" can also be used for cell populations comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker CD56.

[0046] As used herein, the term "pluripotent" refers to the differentiation potential of mesenchymal cells characterized by in vitro differentiation potential towards at least the adipogenic, chondrogenic and osteogenic lineages.

[0047] As used herein, the term "oligopotent" refers to the differentiation potential of mesenchymal cells characterized by in vitro differentiation potential limited to myogenic lineages such as smooth muscle, striated muscle and cardiac muscle.

[0048] As used herein, the term "mesenchymal cell" refers to cells that are positive for CD105, CD90 and CD73 and negative for CD14, CD19, CD34, CD45 and HLA-DR (MHCII).

[0049] As used herein, the term "CD34+" or "CD34 positive" refers to cells that express the cell marker CD34. The term "CD34+" or "CD34 positive" can also be used for cell populations comprising different cell types if preferably at least 80, 90, 95, 98 or 99% of the cell population expresses the cell marker CD56.

[0050] As used herein, the term "CD34-" or "CD34 negative" refers to cells that do not express the cell marker CD34. The term "CD34-" or "CD34 negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0% of the cell population expresses the cell marker CD34. In certain preferred embodiments, the term "CD34-" or "CD34 negative" can also be used for a cell population comprising different cell types if preferably at most 19, 10, 5, 4, 3, 2, 1, or 0% of the cell population expresses the cell marker CD34.

[0051] As used herein, the term "CD146+" or "CD146 positive" refers to cells that express the cell marker CD146. The term "CD146+" or "CD146 positive" can also be used for a cell population 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 CD146.

[0052] As used herein, the term "CD146-" or "CD146 negative" refers to cells that do not express the cell marker CD146. The term "CD146-" or "CD146 negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0% of the cell population expresses the cell marker CD146.

[0053] As used herein, the term "CD49a+" or "CD49a positive" refers to cells that express the cell marker CD49a. The term "CD49a+" or "CD49a positive" can also be used for a cell population 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 CD146.

[0054] As used herein, the term "CD49a-" or "CD49a negative" refers to cells that do not express the cell marker CD49a. The term "CD49a-" or "CD49a negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0% of the cell population expresses the cell marker CD49a.

[0055] As used herein, the term "CD73+" or "CD73 positive" refers to cells that express the cell marker CD73. The term "CD73+" or "CD73 positive" can also be used for a cell population 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 CD73.

[0056] As used herein, the term "CD73-" or "CD73 negative" refers to cells that do not express the cell marker CD73. The term "CD73-" or "CD73 negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker CD73.

[0057] As used herein, the term "CD90+" or "CD90 positive" refers to cells that express the cell marker CD90. The term "CD90+" or "CD90 positive" can also be used for a cell population 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 CD90.

[0058] As used herein, the term "CD90-" or "CD90 negative" refers to cells that do not express the cell marker CD90. The term "CD90-" or "CD90 negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker CD90.

[0059] As used herein, the term "CD105+" or "CD105 positive" refers to cells that express the cell marker CD105. The term "CD105+" or "CD105 positive" can also be used for a cell population 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.

[0060] As used herein, the term "CD105-" or "CD105 negative" refers to cells that do not express the cell marker CD105. The term "CD105-" or "CD105 negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker CD105.

[0061] As used herein, the term "aSMA+", or "aSMA positive", refers to cells that express the cell marker aSMA. The term "aSMA+", or "aSMA positive", can also be used for a cell population 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 aSMA.

[0062] As used herein, the term "aSMA-", or "aSMA negative", refers to cells that do not express the cell marker aSMA. The term "aSMA-", or "aSMA negative", can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker aSMA.

[0063] As used herein, the term "desmin positive", or "desmin+", refers to cells that express the cell marker desmin. The term "desmin positive" can also be used for a cell population 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.

[0064] As used herein, the term "desmin negative", or "desmin-", refers to cells that do not express the cell marker desmin. The term "desmin negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker desmin.

[0065] As used herein, the term "smooth muscle protein positive", or "smooth muscle protein+", refers to cells that express the cell marker smooth muscle protein. The term "smooth muscle protein positive" can also be used for a cell population 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 smooth muscle protein.

[0066] As used herein, the term "smooth muscle protein negative", or "smooth muscle protein-", refers to cells that do not express the cell marker smooth muscle protein. The term "smooth muscle protein negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker smooth muscle protein.

[0067] As used herein, the term "fusion-competent", or "skeletal muscle-derived", refers to cells that are capable of fusing within multinucleated myotubes after being cultured in a skeletal muscle differentiation medium for 5 - 7 days, wherein the multinucleated myotubes have at least 50, 60, 70, 80, 90 or 100% nuclei.

[0068] As used herein, the term "non-fusogenic" or "non-skeletal muscle-derived" refers to cells that cannot fuse with multinucleated myotubes after being cultured in skeletal muscle differentiation medium for 5 - 7 days, where the multinucleated myotubes have less than 50% or at most 49, 30, 20, 10, or 0% nuclei.

[0069] As used herein, the term "skeletal muscle differentiation medium" refers to a cell culture medium that induces fusion into cells such as myoblasts in multinucleated fusogenic cells or myogenic cells. However, if the multinucleated fusogenic cells or myogenic cells can fuse without the corresponding induction, the term also refers to a cell culture medium that does not contain any substances necessary for inducing fusion.

[0070] As used herein, the term "smooth muscle differentiation medium" refers to a cell culture medium that induces cells to transdifferentiate into a smooth muscle phenotype. However, if the cells can transdifferentiate without the corresponding induction, the term also refers to a cell culture medium that does not contain any substances necessary for inducing transdifferentiation.

[0071] As used herein, the term "cell growth medium" refers to any medium suitable for culturing mammalian cells such as SMDC, which allows the attachment of the mammalian cells to the surface of a culture vessel and their proliferation.

[0072] As used herein, the term "contraction" means that the collagen gel lattice contracts by at least 40% from the initial gel size within 48 hours.

[0073] As used herein, the term "non-contraction" means that the collagen gel lattice contracts by less than 40% from the initial gel size within 48 hours.

[0074] The term "TGF-β" is used for transforming growth factor β, which is a multifunctional cytokine belonging to the transforming growth factor superfamily, including three different subtypes (TGF-β1, 2, and 3) and many other signaling proteins produced by all leukocyte lineages. The term "TGF-β" is used synonymously with the terms "TGF-β", "TGF-b", "TGFb", and "TGFB".

[0075] As used herein, the term "AChE positive" or "AChE+" refers to an acetylcholinesterase enzyme activity of at least 1x10 3 mUrel / mg cell protein measured in cells that have been cultured in a smooth muscle differentiation medium as described, for example, in the examples herein. Alternatively, the acetylcholinesterase enzyme activity can be measured by any test known in the art, as described, for example, by Thurner et al., 2018.

[0076] As used herein, the term "AChE negative" or "AChE-" refers to acetylcholinesterase enzyme activity measured in cells that have been cultured in a smooth muscle differentiation medium as described, for example, in the examples herein, which is less than 1x10 3 mUrel / mg cellular protein. Alternatively, acetylcholinesterase enzyme activity can be measured by any test known in the art, as described, for example, by Thurner et al., 2018.

[0077] As used herein, the term "CK positive" or "CK+" refers to creatine kinase activity measured in cells that have been cultured in a smooth muscle differentiation medium as described, for example, in the examples herein, which is at least 1x10 2 mU rel / mg cellular protein. Alternatively, creatine kinase activity can be measured by any test known in the art, as described, for example, by Thurner et al., 2018.

[0078] As used herein, the term "CK negative" or "CK-" refers to creatine kinase activity measured in cells that have been cultured in a smooth muscle differentiation medium as described, for example, in the examples herein, which is less than 1x10 2 mUrel / mg cellular protein. Alternatively, creatine kinase activity can be measured by any test known in the art, as described or analyzed by one of ordinary skill in the art (Thurner et al., 2018).

[0079] As used herein, the term "CD49e+" or "CD49 positive" refers to cells that express the cell marker CD49e. The term "CD49e+" or "CD49e positive" can also be used for a cell population 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 CD49e.

[0080] As used herein, the term "CD49e-" or "CD49e negative" refers to cells that do not express the cell marker CD49e. The term "CD49e-" or "CD49e negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker CD49e.

[0081] As used herein, the term "Pax-7+" or "Pax-7 positive" refers to cells that express the transcription factor Pax-7. The term "Pax-7+" or "Pax-7 positive" can also be used for a cell population 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 Pax-7.

[0082] As used herein, the term "Pax-7-" or "Pax-7 negative" refers to cells that do not express the cell marker Pax-7. The term "Pax-7-" or "Pax-7 negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker Pax-7.

[0083] As used herein, the term "SSEA4+" or "SSEA4 positive" refers to cells that express the cell surface marker SSEA4. The term "SSEA4+" or "SSEA4 positive" can also be used for a cell population 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 SSEA4.

[0084] As used herein, the term "SSEA4-" or "SSEA4 negative" refers to cells that do not express the cell surface marker SSEA4. The term "SSEA4-" or "SSEA4 negative" can also be used for a cell population comprising different cell types if preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0% of the cell population expresses the cell marker SSEA4.

[0085] As used herein, the term "MPC" refers to myogenic progenitor cells. In particular, the term "MPC" refers to myogenic progenitor cells characterized by negative expression of aSMA, CD49a and CD146. MPCs are of skeletal muscle origin and non-pluripotent. MPCs are also characterized by positive expression of CD105, CD90, CD73, CD56, desmin, ACHE and / or CK and / or negative expression of CD34. Examples of the characteristics of MPCs are shown in Figure 15 shown.

[0086] As used herein, the term "MSC" refers to mesenchymal stromal cells. In particular, the term "MSC" or "MSCs" refers to mesenchymal stromal cells characterized by negative expression of aSMA, CD49a and CD146. Mesenchymal stem cells are non-fusogenic and non-skeletal muscle-derived but pluripotent. MSCs are also characterized by positive expression of CD105, CD90, CD73 and / or desmin and / or negative expression of CD56, CD34, desmin, ACHE and / or CK. Examples of the characteristics of MSCs are shown in Figure 15 shown.

[0087] According to the present invention, there is provided a method for obtaining induced smooth muscle cells (iSMCs) from skeletal muscle-derived cells (SMDCs).

[0088] Skeletal muscle cell-derived induced smooth muscle cells

[0089] The first subject matter of the present invention relates to a method for obtaining induced smooth muscle cells (iSMCs), the method comprising the steps of: (a) obtaining skeletal muscle-derived cells from a subject; (b) transdifferentiating the skeletal muscle-derived cells by culturing the cells in a culture medium containing TGF-β, in particular TGFβ1, TGFβ2 and / or TGFβ3, and heparin to obtain iSMCs. In a particularly preferred embodiment, in step (b), the skeletal muscle-derived cells are transdifferentiated by culturing the cells in a culture medium containing TGFβ1 and / or TGFβ3, more preferably TGFβ1, and heparin to obtain iSMCs. Step (b) of the present invention is carried out in vitro or ex vivo. Thus, the method according to the present invention is in vitro or ex vivo.

[0090] In a preferred embodiment of the present invention, step (b) is carried out in a cell culture medium containing 1-10 μg / ml TGFβ1 and 10-30 μg / ml heparin or 1-6 U / ml heparin.

[0091] In a preferred embodiment, the iSMCs obtained according to the method of the present invention, preferably the iSMCs obtained in step (b) of the method according to the present invention, are characterized by positive expression of aSMA, CD49a and CD146.

[0092] In a preferred embodiment of the present invention, the skeletal muscle-derived cells are myogenic progenitor cells (MPCs), which are characterized by positive expression of CD56 and desmin and negative expression of CD34; alternatively, the skeletal muscle-derived cells are mesenchymal stromal cells (MSCs), which are characterized by positive expression of CD105, CD73 and negative expression of CD34 and CD56.

[0093] In a preferred embodiment of the present invention, the skeletal muscle-derived cells are oligopotent MPCs.

[0094] In another preferred embodiment of the present invention, the skeletal muscle-derived cells are MSCs characterized by negative expression of desmin and / or positive expression of CD90.

[0095] In another preferred embodiment of the present invention, the skeletal muscle-derived cells are multipotent MSCs.

[0096] Preferably, in the method according to the present invention, it is foreseen that the iSMCs obtained from MPCs in step (b) are characterized by positive expression of aSMA, CD49a, desmin, CD56 and CD146 and negative expression of CD34; and it is foreseen that the iSMCs obtained from MSCs in step (b) are characterized by positive expression of aSMA, CD49a and CD146 and negative expression of CD56.

[0097] In a further preferred embodiment of the present invention, the iSMC obtained from MPC in step (b) is further characterized by positive expression of smooth muscle proteins.

[0098] In another preferred embodiment of the present invention, the iSMC obtained from MSC in step (b) is further characterized by negative expression of desmin and / or CD34.

[0099] CD73 or 5'-nucleotidase (5'-NT), also known as ecto-5'-nucleotidase, is an enzyme encoded by the NT5E gene in humans. CD73 is commonly used to convert AMP to adenosine. CD73 is expressed on lymphocytes, fibroblasts, smooth muscle cells, endothelial cells, and myoblasts. According to the minimal criteria for MSCs recommended by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006), CD73 is a marker for multipotent mesenchymal stromal cells (MSCs).

[0100] CD105, also known as endoglin, is a type I integral membrane homodimeric protein with 90 kD subunits that is present on vascular endothelial cells and syncytiotrophoblasts of the placenta. CD105 is weakly expressed on stromal fibroblasts. It is also expressed on activated monocytes and tissue macrophages. Expression of CD105 is increased on activated endothelium in tissues undergoing angiogenesis, such as in tumors, or in the context of wound healing or skin inflammation. CD105 is a component of the TGF-β receptor system in human umbilical vein endothelial cells and binds TGF-β1 and β3 with high affinity. According to the minimal criteria for MSCs recommended by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006), CD105 is a marker for multipotent mesenchymal stromal cells (MSCs). Since the present invention contemplates the isolation of iSMC from MPC and / or MSC by incubating MPC and / or MSC with TGFb, CD105 expression may contribute to the success of the methods described herein due to its role as a TGFb coreceptor. Accordingly, the present invention discloses the isolation of CD105-positive MSCs or MPCs (Example 1)( Figure 2 ) and thus can be used to isolate iSMC (Example 2).

[0101] CD34 expression has been described in muscle-derived stem cells and quiescent satellite cells (Qu-Petersen et al., 2002). In addition, CD34-positive skeletal muscle-derived cells have shown enhanced dystrophin regeneration in dystrophic skeletal muscle (Jankowski et al., 2002). The prior art is to generate smooth muscle cells in vitro using CD34+ skeletal muscle-derived cells and to enhance smooth muscle using CD34+ skeletal muscle-derived cells (Capelli et al., 2002). However, normal endogenous smooth muscle is usually CD34-negative (https: / / www.proteinatlas.org / ENSG00000174059-CD34 / tissue / primary+data), but smooth muscle in a neoplastic state often becomes CD34+ (van de Rijn et al., 1994). Therefore, cells lacking CD34 obtained by the method of the present invention may be beneficial for smooth muscle regeneration and are less likely to undergo malignant transformation. According to the method of the present invention, CD34-negative skeletal muscle-derived cells are obtained in the first step and differentiated into CD34-negative iSMCs in the second step (Examples 1 and 2).

[0102] CD146 is a surface protein and receptor for laminin α4 and is present in the extracellular matrix of developing smooth muscle tissue (Iivanainen et al., 1995). In addition, CD146 has been shown to be expressed in bone marrow-derived stem cells committed to the smooth muscle lineage (Espagnolle et al., 2014). As Figure 2 shown, bladder-derived smooth muscle cells are positive for CD146. Summarizing, CD146+ cells labeling the smooth muscle committed cell population are preferred for use in smooth muscle regeneration and / or tissue engineering. This method allows the isolation of CD146+ iSMCs from skeletal muscle-derived cells.

[0103] 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 in smooth muscle tissue in vivo (Romanska et al., 1996). CD56 is present in desmin+ SMDCs with fusion ability (referred to herein as MPCs) and iSMCs derived from the latter. CD56 is the main distinguishing marker between MPCs and skeletal muscle-derived MSCs described herein, since both MSCs and iSMCs derived from the latter are CD56-negative. CD56+ iSMCs obtained as in the present invention may be most suitable for smooth muscle regeneration.

[0104] α-smooth muscle actin (aSMA) is one of the first markers of smooth muscle commitment during development (McHugh, 1995). Its presence is essential for function and contractility due to force transduction in smooth muscle cells (J. Wang et al., 2006). Thus, it is essential to use aSMA as a marker for iSMCs for the regenerative use of smooth muscle function. This method (Example 2) allows the generation of aSMA+ cells from skeletal muscle-derived cells ( Figure 3 ).

[0105] Desmin is one of the earliest known myogenic markers present in all muscle types. Lack of desmin leads to muscle degeneration and dysfunction (Capetanaki et al., 1997). Thus, the use of desmin+ cells for muscle regeneration should be preferred. As Figure 3 shown, iSMCs derived from MPCs are desmin positive.

[0106] CD49a or integrin α1 protein (also known as VLA-1), produced by the expression and translation of the ITGA1 gene, is present during smooth muscle development, particularly in smooth muscle tissues such as the aorta (Belkin et al., 1990). This method allows the isolation of CD49a positive iSMCs from CD49a negative MPCs or MSCs. As Figure 2 depicted, human bladder-derived smooth muscle cells (hBd-SMCs) are also CD49a positive.

[0107] The functionality of smooth muscle tissue depends on the presence of highly differentiated smooth muscle cells that express contractile proteins such as smooth muscle proteins (Niessen et al., 2005). Additionally, smooth muscle proteins are well-known markers of fully differentiated smooth muscle cells and are the first markers to disappear upon smooth muscle injury (van Eys et al., 2007). Thus, the use of smooth muscle protein+ cells in smooth muscle tissue regeneration may be advantageous. This method (Examples 2 and 5) allows the isolation of smooth muscle protein+ iSMCs from smooth muscle protein- MPCs ( Figure 3 ).

[0108] As mentioned above, the markers CD146, CD56, aSMA, CD34, desmin, CD49a, smooth muscle protein are important for the identification and / or function of smooth muscle cells, and a combination of markers may be beneficial for the identification of iSMCs suitable for smooth muscle regeneration. The method presented herein allows the isolation of iSMCs using a combination of the proposed markers (Example 2).

[0109] Specifically, the present invention provides a method for obtaining induced smooth muscle cells (iSMC) from muscle progenitor cells (MPC) derived from skeletal muscle. The iSMC obtained from MPC are 56+, aSMA+, CD49a+, desmin+, CD146+ and CD34-. The latter markers as described above are advantageous for identifying smooth muscle cells because, for example, hBd-SMC are positive for CD146 and CD49a ( Figure 2 ), and are thus preferred for cells to be used as smooth muscle regenerative cells. The combination of CD56+, aSMA+, CD49a+, desmin+, CD146+ and CD34- marker expression is advantageous and novel for iSMC generated in vitro from MPC derived from human skeletal muscle.

[0110] In addition, the present invention provides a method for obtaining iSMC from mesenchymal stem cells (MSC) derived from skeletal muscle. The iSMC derived from skeletal muscle MSC are aSMA+, CD146+, CD49a+, CD56-, and preferably also desmin- and / or CD34-. The positive expression of aSMA, CD49a and CD146 and the negative expression of CD56 and preferably also CD34 are suitable for identifying these cells as MSC-derived smooth muscle cells and are related to the function of iSMC as smooth muscle regenerative cells. The combination of aSMA+, CD146+, CD49a+ and CD56- marker expression on iSMC from skeletal muscle-derived MSC is advantageous and novel compared to previous methods in the art.

[0111] In another preferred embodiment of the present invention, the method includes performing step (a1) after step (a), including proliferating skeletal muscle-derived cells, preferably to obtain 20 - 40x10 6 cells.

[0112] In a particularly preferred embodiment of the present invention, skeletal muscle-derived cells are proliferated to obtain 50x10 6 cells.

[0113] In another preferred embodiment of the present invention, step (b) is performed for 1 to 6 days. In a particularly preferred embodiment of the present invention, step (b) is performed for 3 to 6 days.

[0114] Another subject of the present invention relates to induced smooth muscle cells (iSMC) obtained by the method according to the present invention.

[0115] In a further preferred embodiment of the present invention, the induced smooth muscle cells (iSMC) obtained from MPC are characterized by the positive expression of aSMA, CD49a, desmin, CD56 and CD146 and the negative expression of CD34.

[0116] As already disclosed above, due to the phenotypic replication of the natural expression profile of smooth muscle cells by the methods described herein, the combination of markers aSMA, CD49a, desmin, CD56, and CD146 present or the marker CD34 absent in iSMCs derived from MPCs is advantageous.

[0117] In another preferred embodiment of the present invention, the induced smooth muscle cells (iSMCs) obtained from MPCs are non-fusogenic.

[0118] The fusion of a single nucleated myogenic progenitor cell (MPC) or other fusogenic muscle-derived cells (e.g., myoblasts) with multinucleated myotubes is a prerequisite for skeletal muscle formation and regeneration (Rochlin et al., 2010). However, since smooth muscle tissue in vivo is not composed of multinucleated myotubes but of differentiated single nucleated smooth muscle cells, non-fusogenic cells have an advantage for the regeneration of smooth muscle. Thus, the present invention anticipates that iSMCs derived from MPCs as well as iSMCs derived from MSCs are non-fusogenic. Although the MPCs obtained as shown in Example 1 of the present invention are fusogenic, the iSMCs obtained and ultimately intended for application to smooth muscle tissue as shown in Example 2 of the present invention are non-fusogenic. This applies to both MPC-iSMCs and MSC-iSMCs according to the present invention, such as Figure 15 shown.

[0119] In a further preferred embodiment of the present invention, the induced smooth muscle cells (iSMCs) obtained from MSCs are characterized by positive expression of aSMA, CD49a, and CD146 and negative expression of CD56.

[0120] Preferably, the iSMCs obtained from MSCs are further characterized by negative expression of desmin and / or CD34.

[0121] Since skeletal muscle-derived MSCs differ from MPCs in terms of CD56, the iSMCs derived from MSCs are also CD56-negative. However, MSC-derived iSMCs are aSMA+, CD146+, and CD34-, which is consistent with the smooth muscle specification of MSC-derived iSMCs representing MPC-derived iSMCs and is thus advantageous. In addition, due to the mesenchymal nature of the desired smooth muscle tissue, positive expression of the mesenchymal markers CD90, CD105, and CD73 in iSMCs derived from MSCs by the present invention is preferred.

[0122] Preferably, the expression of the various markers as described above is tested in vitro. In addition, the expression of the various markers as defined above refers to their expression in the respective cells in vitro. In a preferred embodiment, the in vitro expression of aSMA and desmin in the respective cells as defined above corresponds to their respective in vivo expression.

[0123] Preferably, the induced smooth muscle cells (iSMC) express functional calcium and / or potassium channels.

[0124] The function of smooth muscle tissue depends on the presence of functional voltage-gated calcium and potassium channels, which enable regulated cell contraction and regulation of membrane potential, respectively (Sanders, 2008). After nerve stimulation, the smooth muscle cell membrane depolarizes, which triggers the opening of voltage-sensing calcium channels and allows calcium ions to enter the cell from the intercellular space (Sanders, 2008). This event then triggers a signaling cascade that ultimately leads to actin / myosin-induced smooth muscle cell contraction, which is required in detail for functions such as the contraction of the internal anal sphincter to hold liquids, gases, and solids from involuntary release from the rectum (Webb, 2003). In addition, after neuron-induced smooth muscle membrane depolarization, voltage-gated potassium channels open to repolarize the membrane to allow further depolarization in the case of subsequent neuronal signals. Thus, the presence of calcium and potassium channels on iSMC is suitable for the identification of functional iSMC in vitro. In fact, functional iSMC are necessary for dysfunctional regeneration, such as the dysfunction of the underperforming internal anal sphincter in patients with fecal incontinence. The present invention allows the generation of iSMC from skeletal muscle-derived cells that have both functional voltage-gated potassium channels and calcium channels, which may be beneficial for their application in smooth muscle regeneration.

[0125] Preferably, the induced smooth muscle cells (iSMC) are contractile in vitro. One of the typical functions of smooth muscle tissue is contraction (Webb, 2003). To test contractility in vitro, cells are seeded on collagen gels, and the reduction in collagen gel size over time is quantified as a measure of contractility. The inventors have found that iSMC derived from MPC and iSMC derived from MSC are contractile compared to their originating MPC and MSC.

[0126] Preferably, according to the method of the present invention, the induced smooth muscle cells (iSMC), particularly MPC-iSMC, obtained preferably in step (b) are CD49e-. CD49e expression is preferably tested in vitro. CD49e, also known as integrin α5, is a cell adhesion molecule that constructs a heterodimeric receptor with integrin β1 for binding fibronectin, fibrinogen, and microfibrillin-1. Since fibronectin inhibitors are sufficient to increase smooth muscle gene expression, fibronectin signaling supported by CD49e may impede smooth muscle expression. The inventors have found that iSMC derived from murine MPC lack CD49e. The lack of CD49e may contribute to reduced fibronectin signaling, and thus the CD49e-iSMC obtained according to the present invention may be superior to cells known in the art for application in smooth muscle regeneration.

[0127] In an alternative preferred embodiment, in the method according to the invention, the induced smooth muscle cells (iSMCs) obtained preferably in step (b), in particular MPC-iSMCs, are CD49e+. CD49e expression is preferably tested in vitro.

[0128] Preferably, in the method according to the invention, the induced smooth muscle cells (iSMCs) obtained preferably in step (b), in particular MPC-iSMCs, are AChE-. AChE expression is preferably tested in vitro. One of the typical functions of skeletal muscle-derived cells is to express the active AChE enzyme during the in vitro fusion process (Thurner et al., 2018), as it is necessary to terminate nerve signals at the neuromuscular endplate of muscle movement. However, smooth muscle is not innervated by motor neurons of muscle movement, so its contraction is mainly not regulated by acetylcholine and does not require AChE to terminate. The inventors have analyzed the AChE activity of the iSMCs isolated according to Example 3 according to Example 17 and found that the iSMCs are AChE-( Figure 14 ).

[0129] Preferably, in the method according to the invention, the induced smooth muscle cells (iSMCs) obtained preferably in step (b), in particular MPC-iSMCs, are CK-. CK expression is preferably tested in vitro. One of the typical functions of skeletal muscle-derived cells is to express the active CK enzyme during the in vitro fusion process (Thurner et al., 2018), as it is necessary for skeletal muscle contraction. However, smooth muscle contraction is not regulated by creatine kinase (CK), so it is not necessary for smooth muscle cells. The inventors found by analyzing CK activity (Example 17) that the iSMCs (Example 3) are CK-( Figure 14 ).

[0130] In a preferred embodiment, in the method according to the invention, the induced smooth muscle cells (iSMCs) obtained preferably in step (b), in particular MPC-iSMCs, are Pax-7 negative, especially if Pax-7 expression is tested in vitro. In a further preferred embodiment, in the method according to the invention, the induced smooth muscle cells (iSMCs) obtained preferably in step (b), in particular MPC-iSMCs, are Pax-7 positive, especially if Pax-7 expression is tested in vitro. Pax-7 is a transcription factor found in cells committed to the skeletal muscle lineage (Krauss et al., 2016). Deletion of Pax-7 in the mouse muscular layer results in a decrease in skeletal muscle and an increase in smooth muscle mass( et al., 2009).

[0131] In a preferred embodiment, the induced smooth muscle cells (iSMCs) obtained in step (b) according to the method of the present invention, particularly MPC-iSMCs, are preferably negative for SSEA4. In another preferred embodiment, the induced smooth muscle cells (iSMCs) obtained in step (b) according to the method of the present invention, particularly MPC-iSMCs, are preferably positive for SSEA4. SSEA4 expression is preferably tested in vitro. SSEA4 is a cell surface marker found in pluripotent stem cells, which are known for their extensive proliferation potential and thus pose a risk of tumorigenesis.

[0132] Treatment based on induced smooth muscle cells

[0133] Another subject matter of the present invention relates to induced smooth muscle cells (iSMCs) for use in a method of treating a disease or disorder of a subject. Preferably, the subject is a human or an animal. In particular, the present invention provides induced smooth muscle cells (iSMCs) for use in a method of treating a human or an animal by surgery or therapy. More specifically, the present invention provides induced smooth muscle cells (iSMCs) for use in cell therapy, particularly for use in smooth muscle cell therapy.

[0134] In another preferred embodiment of the present invention, the disease or disorder is a smooth muscle defect. Preferably, the smooth muscle defect is selected from the group consisting of anal incontinence, urinary incontinence, reflux disease, gastroparesis, overactive and underactive bladder.

[0135] In a particularly preferred embodiment of the present invention, the disease or disorder is fecal incontinence, particularly passive fecal incontinence. Accordingly, the present invention also relates to iSMCs for use in a method of treating anal incontinence, urinary incontinence, reflux disease, gastroparesis, overactive and underactive bladder, particularly fecal incontinence, more particularly passive fecal incontinence.

[0136] Preferably, the iSMCs are injected into the smooth muscle tissue of a subject in need thereof. Preferably, the iSMCs are injected in an amount effective to treat the smooth muscle defect. During preclinical and clinical trials, the skilled person can readily determine the effective amount of the compound to be administered by methods familiar to physicians and clinicians.

[0137] Regeneration of smooth muscle tissue required through cell administration, such as attenuated, atrophied or damaged smooth muscle, such as sphincters, such as the internal anal, internal urethral, lower or upper esophageal sphincters requires local administration of cells into the tissue in need. Local administration of iSMCs can regenerate smooth muscle by implanting the injected iSMCs at the site of administration. The prior art (Chancellor et al., 2001) discloses skeletal muscle-derived cells characterized by desmin +, CD34 + and Blc-2 + and their application in enhancing soft tissues, such as smooth muscle, in, for example, the bladder or anal sphincter. In contrast, the present invention discloses the isolation in a first step of CD34-, CD56 +, desmin + MPCs or pluripotent CD34-, CD56-, CD73 + and CD105 + MSCs skeletal muscle-derived cells. After treatment with TGFb1 and heparin in a second step, iSMCs can be isolated from said cells of skeletal muscle. Thus, these iSMCs acquire a smooth muscle phenotype by expressing smooth muscle markers such as aSMA and CD146 and can thus be beneficial for smooth muscle regeneration. Preferably, these iSMCs are applied to a method of treating a subject by injection into a subject in need. The inventors have found that iSMCs obtained by the method of the present invention administered into the smooth muscle tissue of the pyloric sphincter implant at the site of injection and integrate into the smooth muscle tissue.

[0138] Preferably, iSMCs are administered into the soft tissue in need by multiple injections.

[0139] It has been shown that multiple injections of SMDCs into skeletal muscle improve cell implantation into muscle (Skuk et al., 2014). The present invention contemplates injecting iSMCs into soft tissues such as, for example, smooth muscle at multiple sites of the same continuum of tissue. In particular, the cells can be administered using multiple needles, each needle injecting a determined number of cells.

[0140] The iSMCs according to the present invention can be administered in the form of a pharmaceutical composition comprising the iSMCs and a pharmaceutically acceptable diluent, excipient or carrier. Thus, the present invention also relates to a pharmaceutical composition comprising the iSMCs according to the present invention and a pharmaceutically acceptable diluent, excipient or carrier.

[0141] In another embodiment of the present invention, iSMCs are used in the manufacture of a medicament for treating fecal incontinence, urinary incontinence, reflux disease, gastroparesis, overactive and underactive bladder, especially fecal incontinence, more especially passive fecal incontinence.

[0142] Tissue engineering

[0143] In another subject matter of the present invention, inducible smooth muscle-derived cells are applied to tissue engineering.

[0144] Smooth muscle tissue typically forms circular / tubular structures in the human body, such as blood vessels and the internal anal sphincter. Using the iSMCs obtained herein to generate circular / tubular structures can be used for in vitro engineering of blood vessels or sphincters. In vitro engineered smooth muscle structures can be used to replace dysfunctional smooth muscle structures, such as the smooth muscle sphincter in patients with fecal incontinence, for example. The inventors have found that the iSMCs obtained by the present invention can reconstruct circular three-dimensional structures similar to the three-dimensional structure of the internal anal sphincter. It was found that the cells within these tissue rings express smooth muscle marker proteins, such as aSMA and desmin. The expression of the contractile proteins aSMA and desmin within the tissue rings obtained by the cells of the present invention is essential for the function of the tissue rings, and thus is beneficial for the application of the tissue rings in tissue replacement.

[0145] In addition, it has been found herein that in vitro engineered sphincters derived from iSMCs have the ultrastructural characteristics of native smooth muscle, such as highly abundant actin structures and dense bodies necessary for force transduction, and thus have the function of smooth muscle structures. In addition, the tissue-engineered sphincters obtained by the present invention allow for the formation of caveolae within the cells. Caveolae are known to be essential for calcium handling, for example, through excitation-contraction, excitation-transcription, and pharmacomechanical coupling (Popescu et al., 2006). Therefore, the iSMCs obtained as by the present invention are promising for applications in tissue engineering.

[0146] Drug screening

[0147] In another aspect of the present invention, iSMCs are applied to drug screening. Before any potentially harmful candidate drugs must be used in animal or human studies, generating smooth muscle structures in vitro using iSMCs may help to test new drugs and their effects on smooth muscle cells in vitro.

[0148] Skeletal muscle-derived cells for generating induced smooth muscle cells

[0149] Another aspect of the present invention relates to the use of skeletal muscle-derived cells in obtaining induced smooth muscle cells (iSMCs).

[0150] Common prior art methods use induced pluripotent stem cells (Dash et al., 2016), adipose-derived multipotent mesenchymal stromal cells (G. Wang et al., 2015), or bone marrow-derived multipotent mesenchymal stromal cells (Espagnolle et al., 2014) to obtain smooth muscle cells in vitro, and their sources are significantly different from the skeletal muscle-derived cells used as the source for obtaining induced smooth muscle cells in the present invention. Disadvantages of prior art cells, such as iPSCs used to obtain smooth muscle cells, have a risk of malignant transformation due to the genetic engineering necessary to obtain iPSCs. Another prior art method describes CD34+ skeletal muscle-derived cells as a source of smooth muscle cells. Thus, the cells are cultured in a differentiation medium for 2 - 4 weeks (Lu et al., 2011). In the present invention, CD34-skeletal muscle-derived cells isolated according to the method of the present invention are used for the first time to obtain iSMCs. The skeletal muscle-derived cells are very suitable for isolating iSMCs because the method of the present invention takes less than 1 week.

[0151] In a preferred embodiment of the present invention, the skeletal muscle-derived cells are oligopotent myogenic progenitor cells (MPCs), which are characterized by positive expression of CD56 and desmin and negative expression of CD34; or wherein the skeletal muscle-derived cells are multipotent mesenchymal stromal cells (MSCs), which are characterized by positive expression of CD105, CD73 and negative expression of CD34 and CD56.

[0152] The following examples illustrate the present invention but are not considered limiting. Examples

[0153] Example 1: Isolation of skeletal muscle-derived cells (SMDC)

[0154] According to the following isolation method, SMDCs were enriched for human myogenic progenitor cells (MPCs), murine myogenic progenitor cells (mMPCs), or human multipotent mesenchymal stromal cells (MSCs).

[0155] Isolation of human skeletal muscle-derived myogenic progenitor cells (MPC)

[0156] Specifically, skeletal muscle biopsies were taken from the pectoralis major or biceps brachii of incontinent patients. To take a biopsy, the skin was first opened through an incision about 1 cm long above the muscle until the pectoralis major fascia was reached. After opening the fascia, 1 cm was removed 3Muscle tissue (biopsy). The biopsy was directly transferred to a biopsy transport medium pre-cooled to approximately 4°C, which consisted of Ham's F10 basal medium supplemented with gentamicin (final concentration of 1 - 5 μg / ml). The biopsy was stored in the biopsy transport medium at 1 - 11°C for approximately 26 hours. Subsequently, the biopsy was transferred to a petri dish containing 1xPBS. The muscle tissue was separated from the connective tissue using sterile forceps and a scalpel. Then, the muscle tissue was transferred to another petri dish containing 1xPBS and cut into pieces of 2 - 3 mm 2 in size. After the additional transfer step as described above, the tissue pieces were further cut into 1 mm pieces. Finally, the pieces were transferred to a centrifuge tube containing 1xPBS and centrifuged at 1300 rpm for 10 minutes. After centrifugation, the supernatant was removed, and the muscle tissue was resuspended in 1xPBS supplemented with 8 μg / ml gentamicin. Then the muscle tissue suspension was cooled to 2 - 8°C and maintained for 48 hours. After cooling, the muscle tissue suspension was centrifuged at 1300 rpm for 10 minutes, then the supernatant was removed, and 2.5 ml of digestion solution was added, which contained 1 - 5 mg / ml collagenase in Ham's F10, 2 - 4% v / v Hepes buffer, 0.1 - 10% v / v fetal bovine serum, and 5 - 10 μg / ml gentamicin. Then the muscle tissue suspension was incubated at 37°C, 5% CO 2 for 6 to 20 hours. Subsequently, 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 Ham's F10, and seeded in a cell culture flask. The SMDC attached to the bottom of the culture flask was further maintained by changing the medium every 3 - 4 days and subcultured after reaching confluence and separation. Subculture was carried out until reaching 1x10 7 to 5x10 7 SMDC.

[0157] Isolation of murine skeletal muscle-derived myogenic progenitor cells (mMPC)

[0158] Mouse MPCs were obtained from skeletal muscle biopsies of Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J, hereafter referred to as TdTomato mice (Jackson Laboratory, Maine, USA). Adult mice were sacrificed by cervical dislocation and then the skin on the back was pinched and peeled off. Next, skeletal muscle was obtained from the longissimus dorsi, gastrocnemius, and tibialis anterior muscles using scissors and a scalpel. The muscle was transferred to a sterile Petri dish and covered with 1xPBS. Then, the remaining connective tissue was removed from the skeletal muscle using forceps and a scalpel and discarded. Then, the muscle tissue was digested using a skeletal muscle dissociation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. To separate myogenic progenitor cells (mMPCs) from non-myogenic SMDCs, a satellite cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the manufacturer's instructions. The collected mMPCs and non-myogenic SMDCs were centrifuged and resuspended in mouse growth medium as described above, which consisted of DMEM / Ham's F12 supplemented with 20% FCS and bFGF. Mouse SMDCs were cultured in collagen-coated culture flasks, which were prepared by covering the surface of the culture flask with collagen I from mouse tails diluted 1:10 in 1xPBS at 37°C for 1 hour. Passage was performed as for human SMDCs. Finally, the cells were detached from the cell culture vessel wall, resuspended in the medium, and used immediately or cryopreserved in liquid nitrogen prior to further use.

[0159] Isolation of human skeletal muscle-derived multipotent mesenchymal stromal cells (MSC)

[0160] MSCs were isolated according to the method of Thurner et al. 2018. First, SMDCs were isolated from muscle biopsies (pectoralis major or latissimus dorsi) and expanded in a cGMP environment. Cells were maintained by standard cell culture methods. Briefly, the cells were cultured in growth medium containing Ham's F-10 basal medium supplemented with 10% FCS (inactivated at 57°C for 40 minutes), bFGF, and gentamicin, and incubated at 37°C, 5% CO 2Incubate at a lower temperature. Replace the growth medium every 2 to 3 days. For subculture and harvesting, wash the cells once with 1xPBS and incubate with 1x trypsin solution at 37°C for 5 minutes. Rinse the cells with the growth medium and centrifuge at 400 x g for 10 minutes. Discard the supernatant and resuspend the pellet in the growth medium. Subsequently, purify the MSCs by magnetic-activated cell sorting (MACS). Therefore, use the human CD56 MicroBeads kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Briefly, after harvesting and counting, centrifuge the cells at 400 x g for 10 minutes, discard the supernatant, and resuspend the cells in 10 mL of MACS buffer. After another centrifugation step (400 x g for 0 minutes), resuspend the pellet in 80 μL of MACS buffer. Subsequently, add 20 μL of magnetic CD56 antibody per 1 x 10 7 cells and incubate at 4°C for 15 minutes. Then, perform cell sorting using a Mini MACS separator and collect the CD56-MSCs in the flow-through. Finally, resuspend the cells in the medium and cryopreserve them in liquid nitrogen or use them immediately before further use.

[0161] Example 2: Transdifferentiation of SMDC into iSMC and its isolation

[0162] Seed the MPCs, murine MPCs, or MSCs obtained in Example 1 onto the culture vessel wall and culture them with the growth medium until approximately 70% confluence. Then, wash the cells once with DMEM / F12 (Thermo Scientific, MA, USA). Next, cover the cells with the smooth muscle differentiation medium, which consists of DMEM / F12 supplemented with recombinant human TGFβ1 (Thermo Scientific, MA, USA), sodium heparin salt from porcine intestinal mucosa (Sigma-Aldrich Co. LLC, MO, USA), heat-inactivated (57°C, 40 minutes) fetal bovine serum (Gibco, Thermo Scientific, MA, USA), and gentamicin (Sandoz GmbH, Tirol, Austria) at final concentrations of 10 ng / ml, 3.84 μg / ml, and 5% (v / v), respectively. Finally, culture the cells in the smooth muscle differentiation medium at 37°C, 5% CO2 for 3 - 6 days. Replace the medium every 3 - 4 days. For isolation, detach the cells from the culture vessel wall and collect them in suspension.

[0163] Example 3: Differentiation potential of SMDC

[0164] For in vitro adipogenesis, chondrogenesis, and osteogenic differentiation, 500,000 cells obtained in Example 1 were seeded into 6-well plates (NUNC, Thermo Scientific, MA, USA) and incubated in growth medium at 37°C, 5% CO 2 Culture for 24 hours. Then, the cells were washed once with 5 ml of DMEM / Ham's F12 and covered with 5 ml of adipogenic, chondrogenic, or osteogenic differentiation medium, respectively. Adipogenic, chondrogenic, and osteogenic differentiation medium were supplemented with StemXVivo Human / Mouse / Rat Adipogenic (R&D Systems Inc., MN, USA, StemXVivo Human Osteogenic Supplement (R&D Systems Inc., MN, USA, or ChondrogenesisSupplement( StemXVivo from Thermo Scientific, MA, USA) TM Osteogenesis / adipogenesis basal medium (R&D Systems Inc., MN, USA) composition. Differentiation medium is also supplemented with gentamicin (Sandoz GmbH, Austria) with a final concentration of 3.83 μg / ml. Cells are cultured in their respective differentiation medium for 14 days, replaced every 2-3 days. After 14 days of culture, the presence of adipocytes, chondrocytes and osteocytes is visualized by staining with Oil Red O (adipocytes), Alcian Blue (chondrocytes) and Alizarin Red S (osteocytes) to assess successful differentiation. Microscope images of multiple independent experiments are quantified by Oil Red O, Alcian Blue and Alizarin Red S staining using the Image J software package. Thus, images are loaded and color channels are separated. The red channel is used for Oil Red O and Alizarin Red S staining, while the blue channel is used for Alcian Blue staining. Background is eliminated by setting a universal threshold, and the average pixel intensity of each field of view is obtained by Image J for quantification. Statistical comparisons were performed by unpaired t-test, with p < 0.05 considered significant (*), and p < 0.01 and p < 0.001 as * or **, respectively. TMSkeletal muscle differentiation was initiated by replacing the growth medium with skeletal muscle cell differentiation medium (500 mL, Promo Cell GmbH, Germany) supplemented with 10 mL of skeletal muscle cell differentiation medium supplement pack (PromoCell GmbH, Germany) and 240 μL of gentamicin (8 mg / mL, Sandoz GmbH, Austria) in cell cultures on Delta Surface plastic plates (Thermo Scientific, MA, USA).

[0165] Under appropriate culture conditions, the MPCs and MSCs obtained in Example 1 were assayed for in vitro differentiation into adipogenic, chondrogenic, osteogenic, and skeletal muscle-derived lineages. Staining of adipocytes, chondrocytes, osteocytes, and myotubes was performed as described above. No Oil Red O- and Alizarin Red S-positive cells were present within MPCs, and only low levels of Alcian Blue cells could be detected. Within MSCs, cells positive for Oil Red O, Alcian Blue, and Alizarin Red S were found after culture in adipogenic, chondrogenic, and osteogenic differentiation media, respectively ( Figure 1 A, B), confirming their enrichment for pluripotent cells and status as multipotent mesenchymal stromal cells, as defined by the International Society for Cellular Therapy (Dominici et al., 2006). Desmin-positive multinucleated myotubes were detected only within MPCs ( Figure 1 A). Quantification of the staining intensity of Oil Red O, Alcian Blue, and Alizarin Red S after in vitro adipogenic, chondrogenic, and osteogenic differentiation and calculation of the fusion index after skeletal muscle-derived differentiation of MSCs and MPCs revealed significantly higher staining intensities for Oil Red O (p = 0.0117), Alcian Blue (p = 0.0020), and Alizarin Red S (p = 0.0012) staining in MSCs compared to MPCs, while a significantly higher fusion index (p = 0.0007) was found in MPCs compared to MSCs ( Figure 1 B). Thus, MPCs are mesenchymal oligopotent cells committed to the myogenic lineage. In addition, MSCs are pluripotent and capable of adipogenic, chondrogenic, and osteogenic differentiation in vitro.

[0166] Example 4: Surface marker expression

[0167] To determine surface marker expression, flow cytometry was performed on a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, the cells obtained in Example 1 were harvested by covering with 1x trypsin at 37 °C for 5 minutes, centrifuged at 400 x g and resuspended in 1x PBS supplemented with 1% FCS. 40,000 cells were resuspended in 195 μl of 1x PBS and 5 μL of CD34-PE, CD56-PE, CD146-PE, IgG1-PE, IgG1-FITC, CD90-PE, CD105-PE (all from Beckman Coulter, CA, USA), CD49a-FITC (Miltenyi Biotec, Germany) or CD73-PE (Becton Dickinson, NJ, USA) was added to a 1.5 mL Eppendorf tube and incubated at 4 °C in the dark for 30 minutes. Subsequently, the cells were washed with 1 mL of PBS, centrifuged at 400 x g for 10 minutes and then resuspended in 195 μL of 1x PBS in a 96-well round bottom plate. Then, 5 μL of the viability dye 7-aminoactinomycin D (Beckman Coulter Inc., France) was added to each reaction and the plate was incubated at room temperature in the dark for 10 minutes. Finally, cell events were acquired using Guava InCyte TM v.2.3 software. Histograms and scatter plots were generated with at least 5000 events at a sample flow rate of 1.8 μL / mL. Positive staining was obtained by comparison with an isotype control set to at least 95% negative or with control (negative) cells.

[0168] To characterize MPCs and MSCs, the presence of mesenchymal stem cell lineage markers (CD105, CD90 and CD73), hematopoietic markers (CD34), myogenic markers (CD56) and smooth muscle lineage markers (CD146) was tested on at least 4 samples from individual patients obtained by the method used in Example 1 and their derived iSMCs obtained by the method used in Example 2. An average % positive cells ≥ 50 was considered positive, while an average < 50% was considered negative. Thus, all cell types were found to be positive for CD105, CD90 and CD73, but negative for CD34( Figure 2 ). In addition, MPCs and their derived iSMCs were CD56+, while MSCs and their derived iSMCs were CD56-.

[0169] Interestingly, the expression of the surface markers CD146 (associated with the vascular smooth muscle commitment of MSCs (Espagnolle et al., 2014)) and CD49a (expressed during smooth muscle development (Belkin et al., 1990)) was negative in both MPCs and MSCs, but positive in their derived iSMCs ( Figure 2 ).

[0170] Example 5: Intracellular marker expression

[0171] Immunofluorescence staining was performed as previously described (Thurner et al., 2018) to directly detect intracellular marker expression on gelatin-coated 24-well plates or glass coverslips placed in 6-well plates. For the fluorescent immunolabeling of α-smooth muscle actin (aSMA), smooth muscle protein, or desmin, the cells were incubated with the respective mouse anti-actin α-smooth muscle (Sigma-Aldrich Co. LLC, MO, USA), mouse anti-smooth muscle protein (Merck Millipore, MA, USA), anti-smooth muscle myosin heavy chain (Merck Millipore, MA, USA), or rabbit anti-desmin (Thermo Scientific, MA, USA) antibodies diluted 1:100 in the blocking medium. Goat anti-mouse Alexa488 or donkey anti-rabbit Alexa547 conjugated secondary antibodies (Thermo Scientific, MA, USA) diluted 1:200 in the blocking medium were used. Nuclear counterstaining was performed by incubating the cells with Hoechst 33342 (Sigma-Aldrich Co. LLC, MO, USA) diluted to a final concentration of 2 μg / mL in PBST (0.1% Triton X-100). The cells were (Merck Millipore, MA, USA) mounted and sealed with a glass coverslip. The staining was compared with (1) the procedure without the primary antibody and (2) cells negative for the test antibody. To quantify the number of positive cells, an overlay of Hoechst and antibody staining was performed, and multiple images of at least three independent cell preparations were analyzed. The total number of cells positive for the antibody staining was divided by the total number of cells (nuclei) evaluated by Hoechst staining. Mean and standard error values were calculated to compare the cells obtained from Examples 1 and 2.

[0172] The expression of intracellular contractile smooth muscle proteins (aSMA, Smothelin) and the general myogenic marker desmin was analyzed by fluorescence immunostaining in the MPCs and MSCs obtained in Example 1 and the iSMCs derived therefrom as described in Example 2. The MPCs and MSCs were aSMA- and smooth muscle protein-. In contrast, the iSMCs isolated from both MSCs and MPCs were found to be aSMA+( Figure 3 ). In addition, the iSMCs isolated from MPCs in Example 2 were found to be smooth muscle protein+. Analysis of intracellular desmin expression revealed that the MSCs and their isolated iSMCs were desmin-. In contrast, both MPCs and iSMCs were desmin+( Figure 3 ).

[0173] Example 6: Gene expression

[0174] Total RNA of 1x10 6 MPCs or MSCs obtained as shown in Example 1 and their derived iSMCs as shown in Example 2 were isolated using the RNEasy kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Sample preparation for microarray hybridization was performed as described in the NuGENOvation Pico SL WTA System V2 and NUGEN Encore Biotin Module guidelines (NuGEN Technologies, Inc, San Carlos, CA, USA). The hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescence signals were measured using an Affymetrix GeneChip Scanner 3000 7G. The fluidics and scanning functions were controlled by the Affymetrix GeneChip Command Console v4.1.3 software. Sample processing was performed at the Affymetrix Service Provider and Core Facility “KFB-Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany).

[0175] By using Affymetrix GeneChip Expression Console v1.4, the probe set signals summarized within the log2 scale were calculated using the RMA algorithm, and the probe set IDs with the highest log2 fold change between MPC and MPC-iSMC were used for subsequent analysis and comparison with the log2 fold change between MSC and MPC-iSMC. An overview of all analyzed genes and their respective log2 fold changes between MSC and MSC-iSMC and between MPC and MPC-iSMC is shown in ( Figure 12 ). A Log2 fold change ≥1 was considered upregulated and thus marked with an asterisk (*). A heat map was generated using Multiple Expression Viewer (MeV3.1.0) software to visualize the log2 fold changes and perform hierarchical clustering and k-means clustering based on the Euclidean distance.

[0176] Microarray analysis was performed to study gene expression changes associated with the isolation of iSMCs in the present invention. Smooth muscle protein (SMTN), calmodulin 1 (CNN1), tropomyosin 1 (TPM1), transgelin (TGLN, SM22), integrin-α-3 (ITGA3), integrin-α-1 (ITGA1, CD49a), vinculin (VCL), and melanoma cell adhesion molecule (MCAM, CD146) (Espagnolle et al., 2014; Miano, 2010; Xie et al., 2011), as well as the myogenic determination gene desmin (DES) (Capetanaki et al., 1997), along with all genes necessary for vascular smooth muscle contraction ("KEGG PATHWAY:Vascular smooth muscle contraction-Homo sapiens (human)", no date) were analyzed in detail. Gene expression changes between MPCs and their derived iSMCs were compared with those between MSCs and their derived iSMCs. Log2 FC of 1 or above and log2FC of -1 or below were considered upregulation and downregulation, respectively. During the differentiation of MPCs into MPC-iSMCs, 20.33% of the 123 tested genes were upregulated, and only 3.25% were downregulated, indicating differentiation into the smooth muscle cell phenotype. Compared with MPCs, the upregulated genes in MPC-iSMCs in the KEGG cluster or known smooth muscle marker genes were PPP1R14A, KCNMB1, PLCB4, ACTG2, ITPR1, ADCY6, CALCRL, KCNMA1, GNA13, CNN1, ADCY2, KCNMB4, GUCY1A3, ARAF, PPP1R12A, MAPK1, CALD1, KCNMB2, PRKACB, ARHGEF11, PPP1R12C, ITPR2, PLCB1, and SMTN( Figure 12 ).

[0177] Log2 FC of 1 or above and log2FC of -1 or below were considered upregulation and downregulation, respectively. During the differentiation of MSCs into MSC-iSMCs, 12.20% of the 123 tested genes were upregulated, and only 3.25% were downregulated, indicating differentiation into the smooth muscle cell phenotype. Compared with MSCs, the upregulated genes in MSC-iSMCs in the KEGG cluster or known smooth muscle marker genes were ACTA2, ACTG2, CALD1, GNAQ, ITPR1, MAPK1, MYL9, KCNMA1, PLCB4, PPP1R14A, PRKCE, CNN1, TPM1, TAGLN, and ITGA1( Figure 12) The results showing upregulated gene expressions of ITGA1 encoding CD49a and SMTN encoding smooth muscle protein in MPC-iSMCs supported our finding that the percentages of CD49a-positive cells and smooth muscle protein-positive cells were increased in MPC-iSMCs compared to MPCs, thus confirming the expression of smooth muscle markers in MPC-iSMCs. The results showing upregulated gene expression of ITGA1 encoding CD49a in MSC-iSMCs supported our finding that the percentage of CD49a-positive cells was increased in MSC-iSMCs compared to MSCs, thus confirming the expression of smooth muscle markers in MPC-iSMCs.

[0178] Although the surface protein expression of CD146 (MCAM) was upregulated in MPC-iSMCs, the upregulation of MCAM gene expression was not found in microarray experiments, indicating post-transcriptional regulation of CD146 expression. In addition, k-means clustering analysis of gene expression with log2 FC changes in MPCs and MSCs during differentiation into MPC-iSMCs and MSC-iSMCs led to the identification of similarly upregulated ( Figure 4 A) and downregulated ( Figure 4 B) genes between MPCs and MSCs when their respective iSMCs were separated. PP1R14A, ACTG2, PLCB4, ITPR1, MAPK1, CNN1, ITGA1, and KCNMA1 were upregulated in both MPCs and MSCs, while PLA2G2A was downregulated in both cell types. In summary, 75.61% of the tested genes were similarly upregulated, downregulated, or neither in MPCs and MSCs during differentiation into iSMCs. Although more upregulated genes and fewer downregulated genes were found in MPCs (20.33% upregulated and 3.25% downregulated) compared to MSCs (12.20% upregulated and 5.69% downregulated), no significant difference was found in the percentages of upregulated or downregulated genes between MPCs and MSCs ( Figure 4 C).

[0179] Example 7: Fusion ability

[0180] The fusion capabilities of MPC and iSMC according to Examples 1 and 2 were evaluated based on their fusion indices (FI), respectively. To determine the fusion index (FI), the cells used for skeletal muscle differentiation induction in Example 3 were washed twice with PBS and fixed with 4% PFA for 10 minutes. Subsequently, the cells were washed 3 times with PBS and stained with 2 μg / mL Hoechst 33342 solution for 20 minutes. For each sample, at least three fields of view were captured during immunofluorescence imaging and overlaid with phase contrast images to allow easy detection of cell nuclei and cell boundaries. After calculating the average of all analyzed fields of view, the fusion index for each captured field of view was calculated by dividing the number of nuclei in the tube by the total number of nuclei in each field of view. Only cells with at least 3 nuclei were considered myotubes. For statistical analysis, at least 3 populations from different patients were analyzed for each group.

[0181] Quantification of FI indicated that significantly more MPCs underwent myogenesis compared to their isolated iSMC in Example 2, indicating a reduced skeletal muscle-derived potential of MPCs after isolation of iSMC from MPCs ( Figure 5 A). In addition, the tubes formed by iSMC contained significantly fewer nuclei than those formed by MPCs ( Figure 5 B). In summary, MPCs exhibited fusion capabilities, while their derived iSMCs were considered non-fusion capable.

[0182] Example 8: Electrophysiology

[0183] According to a previously published protocol (Park et al., 2013), patch-clamp analysis was performed on MPC and iSMC obtained according to Example 1 and 2, respectively, with minor experimental details adjusted. The procedure was as follows. Electrophysiological recordings were made in the whole-cell configuration using an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City). Patch pipettes with a resistance of 1 to 4 MΩ were made of borosilicate glass (GC150F-7.5, Clark Electromedical Instruments, UK) and filled with pipette solution. All data were digitized using a DIGIDATA1200 interface (Axon Instruments, Foster City), smoothed by a four-pole Bessel filter and saved to disk. Current traces were sampled at 10 kHz and filtered at 2 kHz. Data acquisition was performed using the pClamp software package (version 10.0, Axon Instruments, Inc.). Analysis was performed using Microcal Origin7.0. Reagents were obtained from Sigma-Aldrich unless otherwise mentioned. Inward currents of voltage-dependent Cav channels were elicited by applying 500-ms depolarizing pulses from a holding potential of -50 to 50 mV. In MPC, MPC-iSMC, and hBd-SMC, superimposed current traces of Kv channels were elicited by stepwise depolarizing pulses from a holding potential of 80 mV in 20-mV steps between -80 and 60 mV.

[0184] It was found that the cells obtained by Example 2 did not exhibit voltage-sensitive inward or outward currents of voltage-dependent calcium channels or voltage-dependent potassium channels. In contrast, the iSMC derived from MPC in Example 2 showed both voltage-sensitive inward and outward currents, as was also found in hBd-SMC ( Figure 6 ). In summary, isolation of iSMC by incubation with TGFb1 and heparin resulted in functional maturation.

[0185] Example 9: Collagen gel lattice contraction

[0186] To measure the contractility of MSC and MPC obtained according to Example 1 and their derived iSMC obtained by Example 2, they were seeded in collagen gel lattices and the percentage reduction in gel size was quantified. Specifically, the medium was removed from sub-confluent cells in a standard cell culture vessel and the cells were washed twice with 1xPBS. The cells were then covered with trypsin and incubated at 37 °C for 5 minutes. After that, the cells were detached by tapping the wall of the culture vessel and resuspended after adding DMEM / Ham's F12 basal medium. The cells were then centrifuged at 400 xg for 10 minutes.

[0187] The supernatant was removed, and the cell pellet was resuspended in DMEM / Ham's F12 to obtain 6x10 5 cells / ml. For each gel, 400 μl of the cell suspension was mixed with 200 μl of a collagen solution from bovine skin (Thermo-Fisher Scientific, MA, USA). Then, 3 μl of 0.1 M NaOH was added, followed by immediate resuspension, and 500 μl of the mixture was transferred to the wells of a 24-well plate (NUNC, Thermo-Fisher Scientific, MA, USA). The 24-well plate was then incubated at 37 °C for 30 minutes to allow gel formation. Then each gel was covered with 500 μl of DMEM / Ham's F12 and released from the bottom of the 24-well plate using a sterile pipette tip to float on the surface. Finally, the gels were incubated at 37 °C, 5% CO 2 2 for 24 hours to allow gel contraction of the included cells. To quantify gel contraction, stereomicroscopic photographs were taken and the gel area was calculated by applying FIJI (image J) software.

[0188] In the collagen gel lattice contraction assay, it was found that MPCs and MSCs isolated according to step (a) of the present invention (Example 1) were non-contractile. In contrast, iSMCs isolated from MPCs and MSCs according to step (b) of the present invention (Example 2) did show significantly higher contractility than the cells originating from (MPCs, MSCs). In summary, compared to the cells isolated in step (a) (Example 1; Figure 7 ) it was found that SMDCs transdifferentiated into iSMCs and isolated according to step (b) (Example 2) were contractile.

[0189] Example 10: Smooth muscle regeneration using iSMC

[0190] To test the potential of iSMCs obtained according to Example 2 in smooth muscle regeneration, iSMCs derived from murine MPCs (obtained according to Example 1) were injected into the pyloric sphincter of adult female SHO-Prkdc scid Hr hr mice. For this, the mice were first anesthetized by intraperitoneal administration of 100 mg / kg ketamine; 10 mg / kg xylazine and 3 mg / kg acepromazine. Ophthalmic ointment was applied during the surgery. The cryopreserved cells were freshly thawed, washed once with 1xPBS and centrifuged at 400 x g for 10 minutes, then the cells were resuspended in 1xPBS to a final concentration of 40 000 000 cells / ml. At the same time, the mice receiving the cells were placed on a heating plate to maintain the body temperature at 37 °C. Then 25 μl of the cell suspension (containing 1 000 000 cells) was mixed with 5 μl Polystyrene beads, 15 μm, mixed with yellow-green or blue (Thermo-Fisher Scientific, MA, USA), which are necessary for tracking the injection site after surgery. To inject iSMCs into the pyloric sphincter, a midline laparotomy was performed, and then the pyloric sphincter region was located, and 30 μl of the cell-Fluosphere mixture was applied using a 28G needle attached to a 1 ml syringe. The peritoneum and the muscle-skin layer were sutured separately using 6-0 Ethicon PDS plus absorbable monofilament continuous sutures. After surgery, 200 mg of Novalgin was applied subcutaneously at / kg body weight for three days. Twelve weeks after cell injection, the mice were sacrificed by cervical dislocation to obtain the pyloric sphincter and image the pyloric sphincter. / kg body weight for three days. Twelve weeks after cell injection, the mice were sacrificed by cervical dislocation to obtain the pyloric sphincter and image the pyloric sphincter.

[0191] According to the manufacturer's instructions, using Living Software version 4.5.2 (PerkinElmer, MA, USA), the freshly isolated pyloric sphincter region was imaged using an IVIS Spectrum (PerkinElmer, MA, USA). Briefly, the pyloric sphincters of the injected SHO mice and the control SHO mice were placed in glass Petri dishes and placed inside the IVIS system. Fluorescent images were taken at a height of 2 cm with an automatic exposure time at the corresponding absorption and emission wavelengths of TdTomato and yellow Fluosphere beads. Subsequently, the signal intensity was adjusted to remove the background signal by comparing with the sphincter explants from control mice.

[0192] For histological analysis, animals were deeply anesthetized with isoflurane and sacrificed by cervical dislocation. Tissues of interest were immediately dissected and cryo-fixed by immersion in liquid nitrogen-cooled 2-methylbutane. Tissues were sectioned at 15 μm on a Leica 1950 cryostat, and sections were collected on Superfrost plus slides and stored at -20 °C before further processing. For immunohistological analysis, sections were fixed with 4% PFA and washed with PBS containing 0.1% Tween-20 (Sigma-Aldrich). Blocking and antibody dilutions were performed using a PBS solution containing 1% bovine serum albumin fraction (Sigma-Aldrich), 0.2% fish skin gelatin (Sigma-Aldrich), and 0.1% Tween-20 (Sigma-Aldrich). After overnight incubation at 4 °C, primary antibodies against tdTomato (Sicgen) or aSMA (Thermo Scientific, MA, USA) were diluted 1:100 in the blocking medium. Secondary antibodies (Thermo Scientific) were diluted 1:500 and applied for 4 h at room temperature. Nuclei were stained with DAPI (Sigma-Aldrich) diluted to a working concentration of 0.5 μg / ml. Sections were then mounted using Prolong Gold Antifade (Life Technologies). Fluorescent images were acquired using an LSM710 confocal microscope and ZEN 2011 Black software (Carl Zeiss).

[0193] The inventors found that iSMCs isolated from MPCs expressing the TdTomato reporter protein obtained from Example 1 according to Example 2 were detectable and co-localized with co-injected fluorescent beads at the pyloric sphincter site 12 weeks after implantation, indicating implantation of iSMCs at the injection site. Histological examination and subsequent fluorescent immunostaining for TdTomato and aSMA showed that TdTomato-positive iSMC cells were found in the circular muscle layer of the pyloric sphincter and in the muscularis mucosa near the co-injected fluorescent beads ( Figure 8 ). TdTomato-positive iSMC cells located within the smooth muscle layer of the pyloric sphincter also expressed the aSMA protein, indicating that they not only transplanted into smooth muscle tissue but also retained the phenotypic characteristics of smooth muscle cells necessary for smooth muscle regeneration after transplantation ( Figure 8 ).

[0194] Example 11: Tissue-engineered smooth muscle

[0195] To test the applicability of the cells obtained by the method of the present text for tissue engineering, 3D cell culture was performed. The cells obtained according to Example 1 or 2 were used, and a method known in the art that allows the generation of a circular sphincter-like structure (Gwyther et al., 2011) was employed. To produce a culture vessel for 3D culture, a template with a circular engraving (inner diameter 4 mm; outer diameter 10 mm) was made of stainless steel to produce a PDMS negative template that could be autoclaved. Next, a 2% (w / v) agarose solution was prepared by weighing 5 g of low-gelling agarose (Sigma-Aldrich, MA, USA) in a flask containing 250 ml of Ham's F10 basal medium. Then, the PDMS template and the agarose solution were autoclaved at 121 °C for 20 minutes. Then, the PDMS template was filled with the agarose solution under sterile conditions and subsequently incubated at room temperature for 1 hour to allow the agarose to solidify. In the next step, the solidified agarose was removed from the PDMS template, individual cubes containing one circular template were cut out from the agarose, and transferred to a 6-well plate containing 2 ml of Ham's F10 basal medium, and then stored at 37 °C until further use. For 3D culture, the cells obtained in Example 1 were centrifuged at 400 xg for 10 minutes, and the cell pellet was resuspended in the medium to reach a concentration of 5 x 10 6 cells / ml. Next, 200 μl of the cell suspension was added to the agarose template, and then incubated at 37 °C for 48 hours without further interference to allow the cells to form a ring. After the incubation time, the medium outside the template in the 6-well plate was discarded, and the 6-well plate was carefully filled with 5 ml of the medium used in Example 1 to produce a 3D culture of MPC or MSC or any of the latter used in Example 2 to produce an iSMC 3D culture. The cells were cultured for 6 days to allow them to mature at 37 °C, 5% CO 2 until analysis. After 6 days of culture, the cells were analyzed by standard light microscopy, cryosectioned, and subsequently immunostained (Example 12) or H&E stained, scanning electron microscopy, or semi / ultrathin sectioned, and subsequently toluidine blue stained or transmission electron microscopy. The MPCs derived from Example 1 that transdifferentiated into iSMCs as described in Example 2 did indeed form a circular sphincter using the method based on Gwyther et al., 2014 above ( Figure 9 ).

[0196] Example 12: Cryosection and immunostaining

[0197] For histological analysis of the circular 3D culture (bioengineered sphincter) obtained according to Example 11, the ring was carefully removed from the agarose mold using a sterile spoon, washed by transfer into an Eppendorf tube containing 1xPBS, and then fixed and permeabilized by immersion in pre-cooled MetOH at -20 °C for 5 minutes. Subsequently, after removing half of the solution, the ring was washed 3 times by diluting the MetOH with 1xPBS. The bioengineered sphincter was cryo-fixed by immersion in liquid nitrogen-cooled 2-methylbutane. Samples were cut at 15 μm on a CM1950 cryostat (Leica, Germany), sections were collected on Superfrost plus slides (Thermo-Fisher Scientific, MA, USA), and kept at -20 °C until further processing. PBS solution containing 1% bovine serum albumin fraction (Sigma-Aldrich Co. LLC, MO, USA), 0.2% fish skin gelatin (Sigma-Aldrich Co. LLC, MO, USA), and 0.1% Tween-20 (Sigma-Aldrich Co. LLC, MO, USA) was used for blocking and antibody dilution. The primary antibody aSMA (Thermo-Fisher Scientific, MA, USA) was diluted 1:100 in the blocking medium and then incubated overnight at 4 °C. The secondary antibody (Thermo-Fisher Scientific, MA, USA) was diluted 1:500 and applied for 4 hours at room temperature. Nuclei were stained with DAPI (Sigma-Aldrich Co. LLC, MO, USA) diluted to a working concentration of 0.5 μg / ml, and actin filaments were stained by incubation with phalloidin (Thermo-Fisher Scientific, MA, USA) diluted 1:100 in PBS for 20 minutes at room temperature. Subsequently, the sections were mounted using Prolong Gold Antifade (Thermo-Fisher Scientific, MA, USA). Fluorescence images were acquired using a Nikon Eclipse TE2000-U inverted microscope.

[0198] It was found that cells within the tissue rings obtained according to Example 10 were positive for α-smooth muscle actin and desmin ( Figure 10 ). However, within the MPC-derived tissue rings, specifically, aSMA was expressed by cells in the outer layer of the ring, while within the tissue rings containing iSMCs, aSMA-expressing cells were distributed throughout the ring. Specifically, it was found that within the rings formed from MPCs that differentiated into iSMCs during 3D culture according to the combination of Examples 2 and 10, more cells expressed aSMA than the cells within the rings formed from MPCs ( Figure 10)。Conversely, desmin was expressed throughout the tissue rings in both MPC- and iSMC-derived tissue rings ( Figure 10 ).

[0199] Example 13: Scanning electron microscopy

[0200] Scanning electron microscopy was performed at the Department of Anatomy and Embryology, the Medical University of Innsbruck, with the help of Angelika and Kristian Pfaller. To analyze the 3D-cultured cells obtained by Examples 1 and 2 (as described in Section 3.2.26) by scanning electron microscopy, the rings were removed from the agarose template, washed once in a 1.5 ml tube with 1xPBS, then fixed with pre-cooled (-20 °C) MetOH, followed by post-fixation in 1% osmium tetroxide for 1 hour, dehydrated with EtOH, and critical point dried in a Bal-Tec CPD 030 critical point dryer (Balzers, Lichtenstein). After (Plano GmbH, Wetzlar, Germany), the samples were mounted on aluminum stubs with electrically conductive carbon cement Leit-C, sputter-coated with 15 nm Au / Pd (Balzers), and examined on a Gemini 982 scanning electron microscope (Carl Zeiss). MPCs (Example 2) obtained according to Example 1 and trans-differentiated into iSMCs during 3D culture as described in Example 2 appeared elongated and were part of the tissue rings on the surface of the tissue rings formed in Example 10 ( Figure 9 ). The cells on the surface of the rings appeared intact and were likely viable ( Figure 9 ).

[0201] Example 14: Transmission electron microscopy

[0202] Transmission electron microscopy was performed on the tissue rings obtained according to Example 10. Therefore, the rings were removed from the agarose template and transferred to a 1.5 ml Eppendorf tube containing 1xPBS for washing. Then, the samples were transferred to a new tube containing 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for fixation and storage (4 °C). After fixation for at least 24 hours, the rings were washed twice with phosphate buffer and then in 1% OsO 4Fix for 45 minutes. Then, wash the rings 3 times with distilled water for 15 minutes each. Next, de-liquidize the rings at increasing EtOH concentrations (70, 80, 90, 100%) for 30 minutes each, then incubate twice in fresh 100% acetone for 20 minutes each. Next, embed the samples in a 2:1 Aceton-Epon mixture for 150 minutes, then incubate overnight in a 1:2 Aceton-Epon mixture. Finally, incubate the rings in pure Epon and rotate on a spinner for 24 hours, changing the Epon after 8 hours. To polymerize the Epon, incubate the rings at 60 °C for 24 hours. Trim the embedded epoxy samples using an Ultratrim (Reichert) and obtain ultrathin sections using an Ultracut S (Reichert). Observe the ultrathin sections at 80 kV using a CM120 TEM (from Philips / FEI) equipped with a MORADA CCD camera (from Olympus / SIS).

[0203] Transmission electron microscopy of the tissue rings obtained according to Example 10 showed that the cells formed caveolae near their plasma membranes ( Figure 11 A and B). It was further found that the cells within the tissue rings had major actin structures (Figure C), forming dense packing structures such as dense bodies at certain parts of the cells (Figure D).

[0204] Example 15: Surface marker expression of murine MPC-iSMC

[0205] To determine the surface marker expression of murine MPC-iSMCs, flow cytometry was performed using a Guava easyCyte6HT 2L flow cytometer (MerckMillipore, Darmstadt, Germany). Briefly, the murine MPC-iSMCs obtained in Example 1 were used for the antibody staining procedure of a commercially available mouse cell surface marker screening panel (BD bioscience, NJ, USA). Thus, according to the manufacturer's instructions, the cells were seeded at 1.25x10 6Cells were suspended in growth medium at a concentration of cells / ml and an aliquot of 100 μl of the cell suspension was mixed with all primary antibodies in a 96-well plate. The mixture was incubated at 4 °C for 30 minutes, then 100 μl of growth medium was added to each well, the plate was centrifuged at 300 x g for 5 minutes, the supernatant was removed, 200 μl of growth medium was added to each well, centrifuged again, the supernatant was removed, and the cells were resuspended in 100 μl of biotinylated secondary antibody (prepared according to the manufacturer's instructions). The cells were incubated again at 4 °C for 30 minutes, then 100 μl of growth medium was added to each well, the plate was centrifuged at 300 x g for 5 minutes, the supernatant was removed, 200 μl of growth medium was added to each well, centrifuged again, the supernatant was removed, and the cells were resuspended in 100 μl of streptavidin conjugated to Alexa647 (prepared according to the manufacturer's instructions). The cells were incubated again at 4 °C for 30 minutes, then 100 μl of growth medium was added to each well, the plate was centrifuged at 300 x g for 5 minutes, the supernatant was removed, 200 μl of growth medium was added to each well, centrifuged again, the supernatant was removed, and the cells were resuspended in 200 μl of Ham's F10 supplemented with 10% FCS. Finally, cell events were acquired using Guava InCyte TM v.2.3 software. Histograms and scatter plots were generated with a sample flow rate of 1.8 μL / mL with at least 5000 events. Positive staining was obtained by comparison with an isotype control set to at least 95% negative or with control (negative) cells. Mouse iSMCs of Example 3 were found to be CD49e-( Figure 13 )

[0206] Example 16: Analysis of intracellular markers by flow cytometry

[0207] Briefly, MPCs and MPC-iSMCs obtained in Example 1 were harvested by covering adherent cells with 1x trypsin at 37 °C for 5 minutes, and the cells were centrifuged at 400 x g after separation. The cells were counted and aliquoted to achieve 50,000 cells / reaction, then centrifuged at 400 x g, and then resuspended in BD Cytofix / Cytoperm fixation and permeabilization solution (BD Biosciences, Pharmingen TM ) and incubated at 4 °C for 20 minutes. Then, BD Perm / wash buffer (diluted 1:10 in distilled water (aqua dest)) (BD Biosciences, Pharmingen TM) Wash the cells and centrifuge. Then resuspend the cells in 1xPBS and incubate with IgG isotype control-Alexa488 (Bioss Antibodies Inc., MA, USA) or anti-Pax-7-Alexa488 (Bioss Antibodies Inc., MA, USA) at 4 °C for 1 hour in the dark. Subsequently, centrifuge the cells, wash with BDPerm / Wash buffer (diluted 1:10 in distilled water), and resuspend in 1xPBS after the final centrifugation step. Acquire cell events by using Guava InCyte TM v.2.3 software. Generate histograms with a sample flow rate of 1.8 μl / ml with a minimum of 3000 events. Obtain the percentage of positive cells by comparing with the isotype control set at 99% negative.

[0208] Example 17: Enzyme activity analysis

[0209] Acetylcholinesterase activity assay

[0210] Reagent and standard preparation:

[0211] Prepare American Public Health Association (APHA) phosphate buffer pH 7.2 (Sigma-Aldrich Co. LLC, Germany) according to the manufacturer's instructions. Briefly, add 17 g of the powder mixture (potassium dihydrogen phosphate 22.66 g / L and sodium carbonate 7.78 g / L) to 400 mL of distilled water. After adding 0.5 mL of Triton X-100, dissolve the mixture with a magnetic stirrer for 30 minutes at room temperature. Make up the final volume to 500 mL with a measuring cylinder and use without further dilution. Store the buffer at 4 °C before use. The Ellman's reagent (5,5'-dithiobis-2-nitrobenzoic acid, DTNB, 0.5 mM) for each AChE assay is freshly prepared by weighing out 2 mg in a 1.5 mL Eppendorf tube. Dissolve it in 1 mL of phosphate buffer (pH 7.2 containing 0.1% triton X-100) by vortexing for 1-2 minutes. Make up the final volume to 10 mL in a 15 mL falcon tube containing phosphate buffer (pH 7.2 containing 0.1% triton X-100) and store at 4 °C before use. Acetylcholine thioiodide (ATI, 5.76 mM) for each AChE assay is freshly prepared by weighing out 2 mg in a 1.5 mL eppendorf tube. Dissolve it in 1.2 mL of distilled water by vortexing for 1-2 minutes and store at 4 °C before use.

[0212] The AChE standard diluent was prepared in phosphate buffer (pH 7.2, containing 0.1% triton X-100) and used immediately. A ready-to-use 50 U / mL AChE stock solution (from Electrophorus electricus) was purchased from AAT Inc., Sunnyvale, CA, USA. According to the manufacturer's instructions, it was diluted to prepare 1000 mU / mL of AChE, and then further diluted at a ratio of 1:2 to obtain 8 different dilutions in the range of 4 - 500 mU / mL.

[0213] Colorimetric measurement:

[0214] To measure the activity of AChE, the cells obtained by culturing in skeletal muscle differentiation medium according to Example 3 were treated as follows: The differentiation medium was carefully removed from the 24-well plate, and immediately 300 μL of 0.5 mM DTNB solution (prepared in phosphate buffer pH 7.2, containing 0.1% triton X-100) was added. After incubation at room temperature in the dark for 2 minutes, 50 μL of 5.76 mM ATI (prepared in distilled water) was added. The reaction contents were incubated at 30 °C in the dark for 60 minutes, and then OD measurement was performed at 412 nm on an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK).

[0215] For AChE enzyme standard analysis: AChE enzyme standard dilutions in the range of 500 to 4 mU / ml were prepared as described above ( Bioquest, Sunnyvale, USA), and 200 μL of each AChE standard enzyme dilution was mixed with 300 μL of 0.5 mM DTNB and 50 μL of 5.76 mM ATI. The OD of the mixture was measured in a 24-well plate for 60 minutes.

[0216] Calculation of AChE mU rel / mg-protein:

[0217] Based on the OD412 value obtained from the 60-minute cell colorimetric measurement, the AChE mU was calculated by extrapolating from the linear standard curve (OD at 412 nm after 7 or 8 minutes) derived from the AChE standard measurement values. rel . Then, the AChE mU rel was divided by the total protein mg of the corresponding cells cultured in skeletal muscle differentiation medium (calculated according to Example 19) to calculate the AChE mU rel / mg protein value.

[0218] Creatine kinase activity measurement

[0219] To measure the activity of AChE, the cells obtained by culturing according to Example 3 in skeletal muscle differentiation medium were processed as follows: The medium of the cells growing on a 24-well plate was gently removed and the cells were washed with 1 ml of Tryrode's salt solution (Sigma-Aldrich Co. LLC, MO, USA). Immediately thereafter, 70 μl of lysis buffer was added directly to the cells. The lysis buffer was prepared by adding 10 μl of Triton-X-100 to 10 ml of dH 2 O (LC-MS-Ultrachromasol, Fluka). After incubation at 4 °C in the dark for 5 minutes, 400 μl of CK-NAC (Thermo Scientific, MA, USA) previously dissolved in 10 ml of dH 2 O was added. In an Anthos Zenith 340rt microplate reader (Biochrom Ltd., Cambridge, UK) set at 30 °C, the reaction was analyzed by OD absorbance measurement at 340 nm. Unless otherwise mentioned, the OD340nm value obtained 21 minutes after the addition of CK-NAC was used for subsequent analysis. According to the manufacturer's instructions, the CK activity was calculated in mU rel and, unless otherwise mentioned, was normalized by dividing it by the mg total protein of the corresponding cells.

[0220] It was found that the MPC of Example 3 for ACHE and CK activity analysis according to this example was AChE+ and CK+, but the iSMC of the present invention (Example 3) was found to be AChE- and CK-( Figure 14 ).

[0221] Example 18: Comparison of the cells of the present invention with cells known in the art

[0222] Thurner, et al. 2018 described the isolation of SMDCs, which were characterized as CD56+ or CD56-. To compare the cells described by Thurner, et al. 2018 with the cells of the present invention, the authors of the mentioned study were contacted and asked to provide samples. The authors agreed to provide the cells isolated according to Thurner et al., 2018, and thus these cells could be tested according to Examples 3, 4, 5, 7, 8, 9, 16, and 17 of the present invention and compared with the MPC, MSC, MPC-iSMC, and MSC-iSMC isolated in Example 1. The results are shown in Figure 15 shown.

[0223] Frudinger et al., 2018 described the isolation of SMDCs, characterized as CD56+. To relate the cells described by Frudinger et al., 2018 to the cells of the present invention, the authors of the mentioned study were contacted and requested to provide samples. The authors agreed to provide the cells isolated according to Frudinger et al., 2018, and thus these cells were tested according to Examples 3, 4, 5, 7, 8, 9, 16 and 17 of the present invention and compared with the MPCs, MSCs, MPC-iSMCs and MSC-iSMCs isolated in Example 1. The results are shown in Figure 15 as follows.

[0224] Example 19: Protein quantification

[0225] According to Thurner et al., 2018, total protein quantification analysis was performed on the cells obtained by culturing in the skeletal muscle differentiation medium according to Example 3. Therefore, the adherent cells were first washed twice with PBS, subsequently covered with PBST (0.1% Triton X-100), and then incubated at room temperature for 10 minutes. Then, the lysate was resuspended and transferred to tubes, vortexed briefly, and then centrifuged at 1200 x g for 4 minutes. Finally, the clarified supernatant was transferred to a new Eppendorf tube, and the protein concentration was determined by measuring the OD at 540 nm using a Pierce BCA Protein Assay Kit (Thermo Scientific, MA, USA) with an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK) according to the manufacturer's instructions.

[0226] References

[0227] Abrahamsson, H. (2007). Gut, 56(6), 877–883.

[0228] Al-Ali, S., Blyth, P., Beatty, S., Duang, A., Parry, B., & Bissett, I. P. (2009). Journal of Anatomy, 215(2), 212–220.

[0229] Bajpai, V. K., Mistriotis, P., Loh, Y.-H., Daley, G. Q., & Andreadis, S. T. (2012). Cardiovascular Research, 96(3), 391–400.

[0230] Belkin, V.M., Belkin, A.M., & Koteliansky, V.E. (1990). The Journal of Cell Biology, 111(5 Pt 1), 2159–2170.

[0231] Bohl, J.L., Zakhem, E., & Bitar, K.N. (2017). Stem Cells Translational Medicine, 6(9), 1795–1802.

[0232] Capelli, C.C., Chancellor, M.B., Huard, J., & Qu, Z. (2002). WO2001078754A3

[0233] Capetanaki, Y., Milner, D.J., & Weitzer, G. (1997). Cell Structure and Function, 22(1), 103–116.

[0234] Chancellor, M.B., Huard, J., Capelli, C.C., & Qu, Z. (2001). WO2001078754A2

[0235] Dash, B.C., Levi, K., Schwan, J., Luo, J., Bartulos, O., Wu, H., Qiu, C., Yi, T., Ren, Y., Campbell, S., Rolle, M.W., & Qyang, Y. (2016). Stem Cell Reports, 7(1), 19–28.

[0236] Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., & Horwitz, E. (2006). Cytotherapy, 8(4), 315–317.

[0237] Espagnolle, N., Guilloton, F., Deschaseaux, F., Gadelorge, M., Sensébé, L., & Bourin, P. (2014). Journal of Cellular and Molecular Medicine, 18(1), 104–114.

[0238] Frudinger, A., D., Schwaiger, W., Pfeifer, J., Paede, J., & Halligan, S. (2010). Gut, 59(1), 55–61.

[0239] Frudinger, A., Pfeifer, J., Paede, J., Kolovetsiou-Kreiner, V., Marksteiner, R., & Halligan, S. (2015). Colorectal Disease: The Official Journal of the Association of Coloproctology of Great Britain and Ireland, 17(9), 794–801.

[0240] Frudinger, Andrea, Marksteiner, R., Pfeifer, J., Margreiter, E., Paede, J., & Thurner, M. (2018). Stem Cell Research & Therapy, 9(1), 233.

[0241] Goode, P.S., Burgio, K.L., Halli, A.D., Jones, R.W., Richter, H.E., Redden, D.T., Baker, P.S., & Allman, R.M. (2005). Journal of the American Geriatrics Society, 53(4), 629–635.

[0242] Huard, J., Yokoyama, T., Pruchnic, R., Qu, Z., Li, Y., Lee, J.Y., Somogyi, G.T., de Groat, W.C., & Chancellor, M.B. (2002). Gene Therapy, 9(23), 1617–1626.

[0243] Iivanainen, A., Sainio, K., Sariola, H., & Tryggvason, K. (1995). FEBS Letters, 365(2–3), 183–188.

[0244] Jung, Y., Bauer, G., & Nolta, J. A. (2012). Stem Cells (Dayton, Ohio), 30(1), 42–47.

[0245] Krauss, R. S., Chihara, D., & Romer, A. I. (2016). Skeletal Muscle, 6.

[0246] Lecourt, S., Marolleau, J.-P., Fromigué, O., Vauchez, K., Andriamanalijaona, R., Ternaux, B., Lacassagne, M.-N., Robert, I., Boumédiene, K., Chéreau, F., Marie, P., Larghéro, J., Fiszman, M., & Vilquin, J.-T. (2010). Experimental Cell Research, 316(15), 2513–2526.

[0247] Li, Y., Wen, Y., Wang, Z., Wei, Y., Wani, P., Green, M., Swaminathan, G., Ramamurthi, A., Pera, R. R., & Chen, B. (2016). STEM CELLS Translational Medicine, 5(12), 1719–

[0248] Lu, S.-H., Lin, A. T. L., Chen, K.-K., Chiang, H. S., & Chang, L. S. (2011). Journal of Cellular and Molecular Medicine, 15(3), 587–592.

[0249] Marolleau, J.-P., Vauchez, K., & Vilquin, J.-T. (2010). EP2206774A1.

[0250] McHugh, K. M. (1995). Developmental Dynamics: An Official Publication of the American Association of Anatomists, 204(3), 278–290.

[0251] Meyer, I., & Richter, H. E. (2015). Women’s Health (London, England), 11(2), 225–238.

[0252] Mimura, T., Kaminishi, M., & Kamm, M. A. (2004). Digestive Surgery, 21(3), 235–241.

[0253] Niessen, P., Rensen, S., Deursen, J. van, Man, J. D., Laet, A. D., Vanderwinden, J.-M., Wedel, T., Baker, D., Doevendans, P., Hofker, M., Gijbels, M., & Eys, G. van. (2005). Gastroenterology, 129(5), 1592–1601.

[0254] Park, W. S., Heo, S. C., Jeon, E. S., Hong, D. H., Son, Y. K., Ko, J.-H., Kim, H. K., Lee, S. Y., Kim, J. H., & Han, J. (2013). American Journal of Physiology. Cell Physiology, 305(4), C377-91.

[0255] Popescu, L. M., Gherghiceanu, M., Mandache, E., & Cretoiu, D. (2006). Journal of Cellular and Molecular Medicine, 10(4), 960–990.

[0256] Quander, C. R., Morris, M. C., Melson, J., Bienias, J. L., & Evans, D. A. (2005). The American Journal of Gastroenterology, 100(4), 905–909.

[0257] Qu-Petersen, Z., Deasy, B., Jankowski, R., Ikezawa, M., Cummins, J., Pruchnic, R., Mytinger, J., Cao, B., Gates, C., Wernig, A., & Huard, J. (2002). The Journal of Cell Biology, 157(5), 851–864.

[0258] Ramkumar, D., & Schulze, K. S. (2005). Neurogastroenterology and Motility: The Official Journal of the European Gastrointestinal Motility Society, 17 Suppl 1, 22–30.

[0259] Rao, S. S. C. (2004). Gastroenterology, 126(1 Suppl 1), S14-22.

[0260] Rochlin, K., Yu, S., Roy, S., & Baylies, M. K. (2010). Developmental Biology, 341(1), 66–83.

[0261] Romanska, H. M., Bishop, A. E., Moscoso, G., Walsh, F. S., Spitz, L., Brereton, R. J., & Polak, J. M. (1996). Journal of Pediatric Gastroenterology and Nutrition, 22(4), 351–358.

[0262] Sanders, K. M. (2008). Neurogastroenterology and Motility: The Official Journal of the European Gastrointestinal Motility Society, 20 Suppl 1, 39–53.

[0263] Sandison, M., & McCarron, J. (2015). The FASEB Journal, 29(1 Supplement), 418.8.

[0264] Skuk, D., Goulet, M., & Tremblay, J. P. (2014). Cell Transplantation, 23(1), 13–25.

[0265] Thurner, M., Asim, F., Garczarczyk-Asim, D., Janke, K., Deutsch, M., Margreiter, E., Troppmair, J., & Marksteiner, R. (2018). PLOS ONE, 13(3), e0194561.

[0266] Vaizey, C. J., Kamm, M. A., & Bartram, C. I. (1997). Lancet (London, England), 349(9052), 612–615.

[0267] van de Rijn, M., Hendrickson, M. R., & Rouse, R. V. (1994). Human Pathology, 25(8), 766–771.

[0268] van Eys, G. J., Niessen, P. M., & Rensen, S. S. (2007). Trends in Cardiovascular Medicine, 17(1), 26–30.

[0269] Wang, G., Jacquet, L., Karamariti, E., & Xu, Q. (2015). The Journal of Physiology, 593(14), 3013–3030.

[0270] Wang, J. Y., & Abbas, M. A. (2013). The Permanente Journal, 17(3), 65–73.

[0271] Wang, J., Zohar, R., & McCulloch, C. A. (2006). Experimental Cell Research, 312(3), 205–214.

[0272] Wang, Y., Han, Z., Song, Y., & Han, Z. C. (2012). Stem Cells International, 2012.

[0273] Webb, R. C. (2003). Advances in Physiology Education, 27(4), 201–206.

[0274] J., Breuer, C., & Neuhuber, W. L. (2009). Developmental Dynamics, 238(4), 864–874.

[0275] Yin, H., Price, F., & Rudnicki, M. A. (2013). Physiological Reviews, 93(1), 23–67.

Claims

1. An in vitro or ex vivo method for obtaining induced smooth muscle cells, the method comprising the following steps: (a) obtaining skeletal muscle-derived cells from a subject; (b) transdifferentiating the skeletal muscle-derived cells by culturing the cells in a medium containing TGF-β and heparin to obtain induced smooth muscle cells, wherein the skeletal muscle-derived cells are myogenic progenitor cells, and the myogenic progenitor cells have the following characteristics: (i) the myogenic progenitor cells are mesenchymal oligopotent cells committed to the myogenic lineage; and (ii) positive expression of CD56 and desmin and negative expression of CD34.

2. The method according to claim 1, wherein the TGF-β in step (b) is TGFβ1, TGFβ2, and / or TGFβ3.

3. The method according to claim 1, wherein the TGF-β in step (b) is TGFβ1 and / or TGFβ3.

4. The method according to claim 1, wherein the TGF-β in step (b) is TGFβ1.

5. The method according to any one of the preceding claims, wherein the induced smooth muscle cells obtained in step (b) are non-fusogenic and / or characterized by positive expression of αSMA, CD49a, and CD146.

6. The method according to any one of the preceding claims, wherein the induced smooth muscle cells obtained from myogenic progenitor cells in step (b) are characterized by positive expression of αSMA, CD49a, desmin, CD56, and CD146 and negative expression of CD34.

7. The method according to any one of the preceding claims, wherein step (a1) is carried out after step (a), and step (a1) comprises proliferating the skeletal muscle-derived cells.

8. The method according to claim 7, wherein step (a1) is carried out to receive 20 - 40x10 6 cells.

9. The method according to any one of the preceding claims, wherein step (b) is carried out for 1 to 6 days.

10. The method according to any one of the preceding claims, wherein step (b) is carried out in a cell culture medium containing 1-10 μg / ml TGFβ1 and 10-30 μg / ml heparin or 1-6 U / ml heparin.

11. Use of skeletal muscle-derived cells for obtaining induced smooth muscle cells, wherein the skeletal muscle-derived cells are myogenic progenitor cells, and the myogenic progenitor cells have the following characteristics: (i) the myogenic progenitor cells are mesenchymal oligopotent cells committed to the myogenic lineage, and (ii) positive expression of CD56 and desmin and negative expression of CD34.

Images