Methods 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 for treating diseases or disorders or for use in tissue engineering, and the use of cells derived from skeletal muscle to obtain iSMCs.

Description

[Technical field]

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

[0002] For example, degeneration of smooth muscle in sphincters can lead to debilitating disorders such as fecal incontinence. Skeletal muscle-derived cells (SMDCs) have been effectively used in the clinic for the regeneration of skeletal muscle sphincters, such as the external anal sphincter and 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 consist of either skeletal muscles, such as the external anal sphincter, or smooth muscles, such as the internal anal sphincter and pyloric sphincter (Al-Ali et al., 2009; Ramkumar & Schulze, 2005). Anal and pyloric sphincter dysfunction are 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 passive fecal incontinence, the main type of fecal incontinence (Vaizey et al., 1997), affecting 78% of all incontinent patients (Mimura et al., 2004). Although not life-threatening, fecal incontinence severely impacts patients' quality of life (Meyer & Richter, 2015), with a prevalence rate of up to 12% in men and women (Goode et al., 2005; Quander et al., 2005). Conservative treatments, such as the application of bulking agents, have limited success in patients with greater severity of incontinence, and surgical approaches are associated with high morbidity and complication rates (JY Wang & Abbas, 2013).

[0004] The function of smooth muscle tissue depends on the presence of highly differentiated smooth muscle cells expressing contractile proteins such as alpha smooth muscle actin (aSMA), desmin, and smoothelin (SMTN) (Capetanaki et al., 1997; van Eys et al., 2007; J. Wang et al., 2006) and also on functional voltage-gated calcium and potassium channels, allowing the induction of regulated cell contraction (Sanders, 2008). The isolation and use of similar cells for the treatment of smooth muscle deficiencies may be a promising therapeutic option. However, currently, no smooth muscle cell therapy is available on the market. The isolation of smooth muscle cells capable of regenerating defective smooth muscle tissue is the first prerequisite to address the clinical use of these cells. The state-of-the-art method to derive smooth muscle cells is the use of smooth muscle tissue as a source. Although these primary smooth muscle cells have been effectively used for smooth muscle regeneration in passive fecal incontinence animal models (Bohl et al., 2017), primary smooth muscle cells are rarely available for autologous transplantation therapy in living humans, are inherently heterogeneous, and may have limited proliferative capacity (Sandison & McCarron, 2015), and therefore hardly qualify as cell therapy candidates for human smooth muscle regeneration. Therefore, we approached the use of highly proliferative stem / progenitor cells that are poised to differentiate into smooth muscle cells.

[0005] Stem / progenitor cells, such as multipotent mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), have been shown to have the potential to transdifferentiate into smooth muscle lineages (Bajpai et al., 2012; Park et al., 2013) and to regenerate smooth muscle in vivo (Li et al., 2016). iPSCs have shown particular promise in smooth muscle differentiation and functionality in vitro (Bajpai et al., 2012), and iPSC-derived smooth muscle progenitor cells have demonstrated the ability to regenerate urinary sphincter in vivo (Li et al., 2016), but safety concerns, such as genetic instability and teratoma formation, limit their utility (Jung et al., 2012). Cellular products derived from adult MSCs have not raised any major health concerns in the majority of clinical trials (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 MSCs 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 for CD56-positive cells, have been shown to improve external anal sphincter weakness associated with fecal incontinence in the clinic (A. Frudinger et al., 2010, 2015; Andrea Frudinger et al., 2018). Furthermore, cells derived from skeletal muscle were found to engraft into the bladder detrusor and improve bladder function (Huard et al., 2002). However, there is limited knowledge regarding SMDCs in terms of smooth muscle cell differentiation and isolation or their regenerative capacity in vivo (Lu et al., 2011), and no studies have evaluated the therapeutic potential of SMDC-derived smooth muscle cells (induced smooth muscle cells) for the regeneration of sphincter smooth muscle.

[0007] Cell therapy approaches for the regeneration of smooth muscle tissue are highly desirable and rely on the use of cells competent for smooth muscle regeneration. In view of the shortcomings of prior art methods, new methods for providing smooth muscle cells are needed.

[0008] Frudinger et al. (2018) teach the isolation of CD56+ skeletal muscle-derived cells, called SMDCs (Andrea Frudinger et al., 2018), as shown in FIG. 6 of Frudinger et al. (2018). The cells are, among others, shown in FIG. 6 of the present disclosure. 15 As shown in (2018), SMDCs are characterized by negative expression of aSMA, CD49a, and CD146a. Furthermore, as described in Frudinger et al. (2018), SMDCs are characterized by positive expression of Pax-7 (Andrea Frudinger et al., 2018). SMDCs described in Frudinger et al. (2018) are myoblastogenic, i.e., they are capable of fusing into multinucleated myotubes.

[0009] EP 2 206 774 A1 relates to a cell population with differentiation potential obtained by isolation from muscle tissue, more particularly skeletal muscle tissue and / or cardiac muscle tissue, preferably endomysium and / or cardiac tissue (Marolleau et al., 2010). The cell population of EP 2 206 774 A1 comprises ALDH positive cells and has in particular myogenic and / or adipogenic and / or osteogenic differentiation potential. In particular, EP 2 206 774 A1 discloses ALDH+ / CD34- cells, ALDH+ / CD34+ cells and SMALD / 34+ cells, all of which are CD146-, as shown in Tables 1, 3 and FIG. 8 of EP 2 206 774 A1. Furthermore, EP 2 206 774A1 discloses SMALD / 34- cells. The above cells are CD146+ and fusion competent as shown in FIG. 3 of EP 2 206 774 A1.

[0010] Lecourt S et al. (2010) have investigated that human skeletal muscle is an essential source of various cell precursors with potential therapeutic prospects. On the one hand, CD56+ cells that are CD49a- and CD49e+ have been described. On the other hand, CD56- cells that are CD146- and SMA- have been described (Lecourt et al., 2010).

[0011] Thurner et al. (2018) disclose the development of an in vitro potency assay for human skeletal muscle-derived cells. In particular, the isolation of both CD56+ and CD56- skeletal muscle-derived cells (SMDCs) is described. 15 As shown in Figure 2a of Thurner et al., both CD56+ and CD56- SMDCs described in Thurner et al. (2018) are aSMA-, CD146- and CD49a-. Furthermore, as shown in Figure 2a of Thurner et al. (2018), CD56+ cells have AChE activity of >1000 mUrel / g. Summary of the Invention

[0012] Underlying the present invention is the technical problem of providing a method for obtaining iSMCs that are safe and effective for use in methods for treating a disease or disorder of interest. A further technical problem underlying the present invention is the provision of cells that are safe and effective for use in the regeneration of smooth muscle tissue.

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

[0014] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1][Figure 1 shows the characterization of skeletal muscle-derived MPCs and MSCs according to their differentiation potential.] The differentiation potential of MPCs and MSCs was assessed after in vitro differentiation into adipogenic, chondrogenic, osteogenic and skeletal myogenic lineages by culturing in the respective differentiation media and detected by Oil Red O (adipocytes), Alcian Blue (chondrocytes), Alizarin Red S (osteocytes) and anti-Desmin / Hoechst (nuclei and myocytes) staining, respectively. Representative images (scale bar = 100 μm) out of at least three individual preparations are shown (A). Quantification of the adipogenic, chondrogenic, osteogenic and skeletal myogenic differentiation potential of MSCs and MPCs by calculation of the mean staining intensity per field of Oil Red O, Alcian Blue or Alizarin Red S staining or by fusion index calculation of at least three individual samples, respectively, is shown (B). Data are expressed as mean ± SD of MPCs and MSCs from at least three individual muscle biopsies. Statistical comparisons were performed by unpaired t test (p<0.05 considered significant). [Diagram 2] [Figure 2 shows the characterization of skeletal muscle-derived MPCs and MSCs and their derived iSMCs by cell surface marker expression.] Surface expression of mesenchymal (CD105, 90, 73), myogenic (CD56), hematopoietic (CD34) and smooth muscle lineage markers (CD146 and CD49a) in skeletal muscle-derived MPCs and MSCs and their derived iSMCs was assessed by flow cytometry from at least three individual human skeletal muscle biopsies, respectively. As a control, expression of smooth muscle lineage markers CD146 and CD49a is shown in a population of human bladder-derived smooth muscle cells (hBd-SMCs). Data are presented as mean ± SEM. [Diagram 3][Figure 3 shows the expression of intracellular markers in SMDCs (MPCs and MSCs) and iSMCs.] Detection of common myogenic (desmin) and smooth muscle myogenic (aSMA, smoothelin) markers in MPCs and MSCs, and in iSMCs derived from them, was performed by immunocytochemistry, and representative images (scale bar = 100 μm) are shown (A). The percentage of cells positive for aSMA, smoothelin, or desmin within MPCs, MSCs, and in iSMCs derived from them, was assessed by quantification of the corresponding marker-expressing cells on immunocytochemistry images of cultures from at least three individual human muscle biopsies (B). Data are presented as mean ± SEM. [Figure 4] [Figure 4 shows gene expression changes during transdifferentiation of SMDCs (MPC and MSC) to iSMCs.] Gene expression changes were assessed by microarray analysis of MSCs and MPCs derived from two individual human muscle biopsies, cultured for 6 days in growth medium (MSC and MPC) or smooth muscle differentiation medium (MSC-iSMC and MPC-iSMC), respectively. Clusters of genes similarly upregulated (A) or downregulated (B) in both MSCs and MPCs upon iSMC differentiation, obtained by k-means, are shown in the heatmap. Asterisks (*) denote genes that were either upregulated (log2 FC ≥ 1) or downregulated (log2 FC ≤ -1) in both cell types. Statistical comparisons were performed by chi-squared tests, with p values ​​less than 0.05 considered significant. Results of gene expression changes are shown in (C). [Diagram 5] [Figure 5 shows the fusion ability of MPCs and iSMCs.] The formation of myotubes (fusion ability) was observed by fluorescence microscopy following Hoechst33342 staining for visualization of nuclei. Based on the images, the fusion index (FI) (A) and the number of nuclei per tube were determined (B). Cells with at least three nuclei were counted as tubes. The measurements were compared between MPCs and their derived iSMCs. [Figure 6][Figure 6 shows the formation of functional ion channels during transdifferentiation of MPC to iSMC.] Analysis of voltage-dependent inward calcium (A) and outward potassium (B) currents in MPC and derived iSMC, and smooth muscle cells derived from bladder (hBd-SMC). Impedance-voltage (IV) curves of at least three cells, MPC, iSMC (derived from MPC), and hBd-SMC, respectively, show the presence of functional Cav (A) and Kv (B) channels in iSMC (derived from MPC) and hBd-SMC, but not in MPC. [Figure 7] [Figure 7 shows the contractility of SMDCs and iSMCs in collagen gel lattices.] The contractility of SMDCs (MSCs and MPCs), iSMCs derived therefrom, and smooth muscle cells derived from bladder (hBd-SMCs) was quantified by collagen gel lattice contraction. The percentage of gel contraction from the original size within 48 hours for cells (MPCs or MSCs) obtained by step (a) of the present invention and cells obtained by transdifferentiation into iSMCs by step (b) of the present invention, as well as control smooth muscle cells derived from human bladder (hBd-SMCs) is shown as a bar graph (A). Data are shown 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 containing embedded MSCs and MSCs and iSMCs derived therefrom, respectively, and hBd-SMCs in wells of a 24-well plate (B). [Figure 8][Figure 8 shows the smooth muscle cell phenotype of iSMCs derived from mMPCs and their engraftment into smooth muscle tissue in vivo.] The percentage of desmin- and aSMA-positive cells in iSMCs derived from mouse MPCs is shown as a bar graph (A). Fluorescent signal detection of TdTomato transgene expressing fluorescent beads and localization in intact pyloric sphincter by in vivo imaging (B). Alpha smooth muscle actin (aSMA) protein expression, TdTomato expression, and overlay (MERGE) of TdTomato and aSMA protein of transplanted iSMCs were detected by immunohistochemistry in pyloric sphincter tissue sections 12 weeks after transplantation. Nuclear counterstaining in each image was performed by DAPI. Representative images of n=8 injected mice are shown (C). [Figure 9] [Figure 9 shows optical and scanning electron microscopy images of tissue rings.] Optical microscopy images of tissue rings obtained by 3D culture of MPC-derived iSMCs arranged around the central post of an agarose template at different magnifications (A and B). Scanning electron microscopy images of tissue rings obtained by 3D culture of MPC-derived iSMCs at different magnifications (C and D). [Figure 10] [Figure 10 shows the expression of contractile proteins in tissue rings derived from MPCs and iSMCs by immunofluorescence.] Immunofluorescence staining for general myogenic (desmin) and smooth muscle myogenic (aSMA) markers and nuclear counterstain (Hoechst) performed on frozen sections of tissue rings obtained from 3D cultures of MPCs and their derived iSMCs. [Figure 11] [Figure 11 shows transmission electron microscopy images of tissue rings.] Images obtained by transmission electron microscopy of ultrathin-sectioned tissue rings obtained by 3D culture of iSMCs show caveolae (arrowheads) on the plasma membrane of two adjacent cells (A and B), abundant filamentous structures in the cytoplasm (C), and dense bodies (arrows) with accumulated filaments (D). [Figure 12][Figure 12 shows quantification of gene expression changes during transdifferentiation of SMDCs (MPC and MSC) to iSMCs.] Gene expression changes were assessed by microarray analysis from MSCs and MPCs to MSC-iSMCs and MPC-iSMCs, which were cultured for 6 days in growth medium (MSC and MPC) or smooth muscle differentiation medium (MSC-iSMC and MPC-iSMC), respectively, and derived from two separate human muscle biopsies. To compare smooth muscle differentiation between MSCs and MPCs, the Log2 fold changes of smooth muscle-related genes in MSC vs. MSC-iSMC (MSC) and MPC vs. MPC-iSMC (MPC) samples are shown. Asterisks (*) indicate genes that are upregulated (Log2 ≥ 1) or downregulated (Log2 ≤ -1). [Figure 13] [Figure 13 shows flow cytometry analysis of anti-CD49e antibody stained and isotype control stained mouse MPC-iSMCs from Example 15.] Dot plots of IgG1 isotype control (A) and anti-CD49e (B) positive MPC-iSMCs show 0.14% control positive cells and 9.5% CD49e positive cells, respectively. [Figure 14] [Figure 14 shows AChE and CK activity analysis of MPC and MPC-iSMC, whereby enzyme activity was measured before and after cell culture in skeletal muscle differentiation medium (SKDiff).] AChE activity (A) and CK activity (B) were measured and compared for MPC and MPC-iSMC, respectively, before and after 6 days of culture in skeletal muscle differentiation medium (SKDiff). Data are presented as mean ± SEM of cells derived from at least three separate human muscle biopsies. Statistical analysis was performed by paired t-test, with p<0.05 considered significant. [Figure 15][Figure 15 shows an overview of the characteristics and marker expression of the different cell types described herein.] CD56+ and CD56- SMDCs (Thurner et al. 2018) and SMDCs (Frudinger et al. 2018) were obtained as described in Example 18. MPCs and MSCs were obtained as described in Example 1. MPC-iSMCs and MSC-iSMCs were obtained as described in Example 2. Expression of a particular marker is indicated as "+" if at least 50% of the cells in the tested cell population expressed the respective cell marker. Expression of a particular marker is indicated as "-" if less than 50% of the cells in the tested cell population expressed the respective cell marker. AChE enzyme activity of a particular cell population is indicated as "+" if the cell population has an AChE activity of at least 1000 mUrel / mg protein measured according to Example 17. The AChE enzyme activity of a particular cell population is indicated as "-" if the cell population has an AChE activity of less than 1000 mUrel / mg protein as measured according to Example 17. The CK enzyme activity of a particular cell population is indicated as "+" if the cell population has a CK activity of at least 100 mUrel / mg protein as measured according to Example 17. The CK enzyme activity of a particular cell population is indicated as "-" if the cell population has a CK activity of less than 100 mUrel / mg protein as measured according to Example 17. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The use of the word "a" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0016] The term "about" means to take into account the stated value, plus or minus 5% of the stated value, or the standard error of measurement of a given value.

[0017] The term "anal incontinence" as used herein refers to the unwanted loss of bowel contents through the anus, such as flatus, liquid or solid stool, etc. The term consists of three severity grades in total: Grade 1 = gas only, Grade 2 = liquid and loose stool, Grade 3 = solid formed stool.

[0018] As used herein, the term "anal sphincter" or "anal sphincter tissue" refers specifically to the external anal sphincter and puborectalis muscles as part of the levator ani, but also includes the pubococcygeus, coccygeus, iliococcygeus and pudendal nerve.

[0019] The term "skeletal muscle-derived cells" or "SMDCs" refers to cells obtained from skeletal muscle tissue, including fusion-competent cells, such as myoblasts, or non-fusion-competent cells, such as multipotent mesenchymal stromal cells, which may be primary cells and / or in vitro cultured cells, or other cells with myogenic or multipotential differentiation potential (e.g., from lipoaspirate tissue or other stem cells, including tissues such as bone marrow). The term also includes cells derived from fat that can be isolated and used for differentiation into smooth muscle cells. The term "skeletal muscle-derived cells" or "SMDCs" also refers to cell populations isolated from muscle tissue.

[0020] The term "human bladder-derived smooth muscle cells (hBd-SMCs)" refers to a population of cells comprising smooth muscle cells from the human bladder. hBd-SMCs are commercially available from PromoCell® (Cat. No. C-12571) and display the phenotypic and functional characteristics of smooth muscle cells predicted to be achieved by iSMCs derived from skeletal muscle-derived cells in the present invention.

[0021] The term "injection" as used herein refers to the ejection of the injection solution containing the above-mentioned cells from the injection device to or adjacent to a specific site in the human body, particularly the muscle tissue that provides anal incontinence. The injection process can be, but is not limited to, static, i.e., the injection device remains at the location where it has arrived. 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.

[0022] The term "injection site" as used herein refers to a site within the human body where the injection process begins, such as near or in muscle tissue that provides anal incontinence. The injection site does not have to be the same as the site where the injection process ends.

[0023] The term "injection device" as used herein refers to any device suitable for penetrating human tissue to reach a desired injection site and capable of delivering a solution, particularly a solution containing muscle-derived cells, to the desired injection site.

[0024] As used herein, the term "fecal incontinence" refers only to the unwanted loss of liquid or formed stool via the anus.

[0025] As used herein, the term "passive incontinence" refers to the lack of sensory awareness of fecal loss. This includes low anal baseline pressure values ​​due to defects in the internal anal sphincter smooth muscle and / or lack of sensory capacity of the anal and rectal mucosa.

[0026] 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 that include different cell types, preferably at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD56.

[0027] 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 that contain different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD56.

[0028] The term "multipotency" as used herein refers to the differentiation potential of mesenchymal cells characterized by in vitro differentiation potential toward at least the adipogenic, chondrogenic, and osteogenic lineages.

[0029] The term "oligopotent" as used herein refers to the differentiation potential of mesenchymal cells characterized by an in vitro differentiation potential that is restricted to the myogenic lineage, such as smooth, striated, and cardiac muscle.

[0030] As used herein, the term "mesenchymal cells" refers to cells that are positive for CD105, CD90, and CD73, and negative for CD14, CD19, CD34, CD45, and HLA-DR (MHCII).

[0031] 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 that include different cell types, preferably where at least 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD56.

[0032] The term "CD34-" or "CD34 negative" as used herein refers to cells that do not express the cell marker CD34. The term "CD34-" or "CD34 negative" can also be used for cell populations that include different cell types, preferably less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD34. In a particularly preferred embodiment, the term "CD34-" or "CD34 negative" can also be used for cell populations that include different cells, preferably at most 19, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD34.

[0033] 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 cell populations that include different cell types, and preferably, at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD146.

[0034] 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 cell populations that contain different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD146.

[0035] 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 cell populations that include different cell types, preferably at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD146.

[0036] As used herein, the term "CD49a-" or "CD49a negative" refers to cells that do not express the cell marker CD49a. The terms "CD49a-" or "CD49a negative" can also be used for cell populations that contain different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD49a.

[0037] As used herein, the term "CD49a-" or "CD49a negative" refers to cells that do not express the cell marker CD49a. The terms "CD49a-" or "CD49a negative" can also be used for cell populations that contain different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD49a.

[0038] 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 cell populations that include different cell types, preferably at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD73.

[0039] 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 cell populations that include different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD73.

[0040] 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 cell populations that include different cell types, preferably at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD90.

[0041] 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 cell populations that contain different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD90. 90 is expressed.

[0042] 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 cell populations that include different cell types, and preferably, at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD105.

[0043] 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 cell populations that contain different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD105.

[0044] 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 cell populations that include different cell types, preferably at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker aSMA.

[0045] 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 cell populations that include different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker aSMA.

[0046] 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 cell populations that include different cell types, preferably at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker desmin.

[0047] 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 cell populations that include different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker desmin.

[0048] As used herein, the term "smoothelin positive" or "smoothelin+" refers to cells that express the cell marker smoothelin. The term "smoothelin positive" can also be used for a cell population that includes different cell types, and preferably, at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker smoothelin.

[0049] The term "smoothelin negative" or "smoothelin-" as used herein refers to cells that do not express the cell marker smoothelin. The term "smoothelin negative" can also be used for a cell population that includes different cell types, and preferably, less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker smoothelin.

[0050] As used herein, the term "fusion competent" or "skeletal myogenesis" refers to cells in which at least 50, 60, 70, 80, 90 or 100 percent of the nuclei within a multinucleated myotube are capable of fusing into multinucleated myotubes after culture in skeletal muscle differentiation medium for 5 to 7 days.

[0051] As used herein, the term "non-fusion competent" or "non-skeletal myogenic" refers to cells in which, after culture in skeletal muscle differentiation medium for 5-7 days, less than 50% or at most 49, 30, 20, 10 or 0 percent of the nuclei within a multinucleated myotube are unable to fuse into multinucleated myotubes.

[0052] As used herein, the term "skeletal muscle differentiation medium" refers to a cell culture medium that induces fusion in multinuclear fusion competent cells or myogenic cells, such as myoblasts. However, the term also refers to a cell culture medium that does not contain substances necessary for induction of fusion, in cases where the multinuclear fusion competent cells or myogenic cells are able to fuse without the respective induction.

[0053] As used herein, the term "smooth muscle differentiation medium" refers to a cell culture medium that induces the transdifferentiation of cells into a smooth muscle phenotype. However, the term also refers to a cell culture medium that does not contain substances necessary for inducing transdifferentiation when the cells are capable of transdifferentiation without the respective induction.

[0054] The term "cell growth medium" as used herein refers to any medium suitable for the incubation of mammalian cells, such as SMDCs, which allows for the attachment of said mammalian cells to the surface of the incubation vessel and their proliferation.

[0055] As used herein, the term "contractile" refers to a collagen gel lattice contraction of at least 40% from the initial gel size within 48 hours.

[0056] The term "non-contractile" as used herein refers to collagen gel lattice contraction of less than 40% from the initial gel size within 48 hours.

[0057] The term "TGF-beta" is used for transforming growth factor beta, a multifunctional cytokine that belongs to the transforming growth factor superfamily, which includes three distinct isoforms (TGF-β1, 2, and 3) and many other signaling proteins produced by all leukocyte lineages. The term "TGF-beta" is used synonymously with the terms "TGF-β," "TGF-b," "TGFb," and "TGFB."

[0058] As used herein, the term "AChE positive" or "AChE+" refers to a cell that is at least 1×10 per mg of cellular protein as measured in cells cultured in smooth muscle differentiation medium, e.g., as described in the Examples herein. 3 It refers to the acetylcholinesterase enzyme activity of mUrel. Alternatively, the acetylcholinesterase enzyme activity can be tested by any test known in the art, for example, as described in Thurner et al., 2018.

[0059] As used herein, the term "CK positive" or "CK+" refers to a CK positive expression level of at least 1×10 CK / mg of cellular protein as measured in cells cultured in smooth muscle differentiation medium, e.g., as described in the Examples herein. 2It refers to the creatine kinase activity of mUrel. Creatine kinase Activity can be tested by any test known in the art, for example, as described in Thurner et al., 2018.

[0060] As used herein, the term "CK negative" or "CK-" refers to a CK-positive cell line that is less than 1×10 CK per mg of cellular protein as measured in cells cultured in smooth muscle differentiation medium, e.g., as described in the Examples herein. 2 It refers to creatine kinase enzyme activity less than mUrel. Alternatively, creatine kinase activity can be tested by any test known in the art, for example, as described or analyzed by one of skill in the art (Thurner et al. 2018).

[0061] As used herein, the term "CD49e+" or "CD49e positive" refers to cells that express the cell marker CD49e. The term "CD49e+" or "CD49e positive" can also be used for cell populations that include different cell types, and preferably, at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD49e.

[0062] 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 cell populations that contain different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD49e.

[0063] 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 cell populations that include different cell types, preferably at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker Pax-7.

[0064] 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 cell populations that include different cell types, preferably where less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker Pax-7.

[0065] 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 cell populations that include different cell types, and preferably, at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker SSEA4.

[0066] 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 that includes different cell types, and preferably, less than 0% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker SSEA4.

[0067] The term "MPC" as used herein 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 skeletal myogenic and not multipotent. MPCs may be further characterized by positive expression of CD105, CD90, CD73, CD56, desmin, ACHE and / or CK, and / or negative expression of CD34. Examples of MPC characteristics are shown in FIG. 15.

[0068] The term "MSC" as used herein refers to mesenchymal stromal cells. In particular, the term "MSC" refers to mesenchymal stromal cells characterized by negative expression of aSMA, CD49a, and CD146. MSCs are non-fusion competent, not skeletal myogenic, but multipotent. MSCs may be further 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 MSC characteristics are shown in FIG. 15.

[0069] According to the present invention, a method is provided for obtaining induced smooth muscle cells (iSMCs) from skeletal muscle derived cells (SMDCs).

[0070] [Smooth muscle cells derived from skeletal muscle cells] A first subject of the present invention relates to a method for obtaining induced smooth muscle cells (iSMCs), comprising the steps of (a) obtaining skeletal muscle-derived cells from a subject, and (b) transdifferentiating the skeletal muscle-derived cells to obtain iSMCs by culturing the cells in a medium containing TGF-beta, in particular TGFb1, TGFb2 and / or TGFb3, and heparin. In a particularly preferred embodiment, to obtain iSMCs, the skeletal muscle-derived cells are transdifferentiated in step (b) by culturing the cells in a medium containing TGFb1 and / or TGFb3, more preferably TGFb1, and heparin. 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.

[0071] In a preferred embodiment of the invention, step (b) is carried out in a cell culture medium containing 1-10 μg / ml TGFb1 and 10-30 μg / ml heparin or 1-6 U / ml heparin.

[0072] In a preferred embodiment, the iSMCs obtained according to the method of the invention, preferably in step (b) of the method according to the invention, are characterized by positive expression of aSMA, CD49a and CD146.

[0073] In a preferred embodiment of the invention, the skeletal muscle-derived cells are myogenic progenitor cells (MPCs) characterized by positive expression of CD56 and desmin, and negative expression of CD34; alternatively, the skeletal muscle-derived cells are mesenchymal stromal cells (MSCs) characterized by positive expression of CD105, CD73, and negative expression of CD34 and CD56.

[0074] In a preferred embodiment of the invention, the skeletal muscle-derived cells are oligopotent MPCs.

[0075] In a further preferred embodiment of the invention, the skeletal muscle-derived cells are MSCs characterized by negative expression of desmin and / or positive expression of CD90.

[0076] In a further preferred embodiment of the invention, the skeletal muscle-derived cells are multipotent MSCs.

[0077] Preferably, in the method according to the invention, the iSMCs obtained from the MPCs in step (b) are expected to be characterized by positive expression of aSMA, CD49a, desmin, CD56 and CD146, and negative expression of CD34, and the iSMCs obtained from the MSCs in step (b) are expected to be characterized by positive expression of aSMA, CD49a and CD146, and negative expression of CD56.

[0078] In a further preferred embodiment of the invention, the iSMCs obtained from the MPCs in step (b) are further characterized by positive expression of smoothelin.

[0079] In a further preferred embodiment of the present invention, the iSMCs obtained from the MSCs in step (b) are further characterized by a negative expression of desmin and / or CD34.

[0080] CD73 or 5'-nucleotidase (5'-NT), also known as ecto-5'-nucleotidase, is an enzyme that in humans is encoded by the NT5E gene. CD73 normally functions to convert AMP to adenosine. CD73 is expressed on lymphocytes, fibroblasts, smooth muscle cells, endothelial cells, and myoblasts. CD73 is a multipotent mesenchymal stromal cell (MSC) marker according to the minimal criteria for MSCs proposed by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006).

[0081] CD105 is also known as endoglin. It is a type I integral membrane homodimeric protein with a subunit of 90 kD found in vascular endothelial cells and in the syncytiotrophoblast of the placenta. CD105 is weakly expressed on stromal fibroblasts. It is also expressed on activated monocytes and tissue macrophages. CD105 expression increases on activated endothelium of tissues undergoing angiogenesis, such as tumors, or in cases of wound healing and inflammation in the skin. CD105 is a component of the TGF-β receptor system on human umbilical vein endothelial cells and binds with high affinity to TGF-β1 and β3. CD105 is a multipotent mesenchymal stromal cell (MSC) marker according to the minimal criteria for MSCs proposed by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006). Since the present invention foresees the isolation of iSMCs from MPCs and / or MSCs by incubating the latter with TGFb, CD105 expression, due to its role as a TGFb coreceptor, may aid in the success of the methods described herein. Thus, the present invention discloses the isolation of MSCs or MPCs (Example 1) that are CD105 positive (Figure 2) and thus useful for the isolation of iSMCs (Example 2).

[0082] CD34 expression was described in muscle-derived stem cells and quiescent satellite cells (Qu-Petersen et al., 2002). Moreover, CD34-positive skeletal muscle-derived cells showed enhanced regeneration of dystrophin in dystrophin skeletal muscle (Jankowski et al., 2002). The state of the art is the use of CD34+ skeletal muscle-derived cells to generate smooth muscle cells in vitro, and for smooth muscle augmentation (Capelli et al., 2002). However, normal endogenous smooth muscle is generally CD34-negative (https: / / www.proteinatlas.org / ENSG00000174059-CD34 / tissue / primary+data), whereas smooth muscle in tumorigenic conditions often becomes CD34+ (van de Rijn et al., 1994). Thus, cells obtained by the method of the present invention that lack CD34 may be favorable for smooth muscle regeneration and less susceptible to malignant transformation. According to the method of the invention, CD34-negative skeletal muscle-derived cells are obtained in a first step and differentiated into CD34-negative iSMCs in a second step (Examples 1 and 2).

[0083] CD146 is a surface protein and receptor for laminin alpha 4, which is present in the extracellular matrix of developing smooth muscle tissue (Iivanainen et al., 1995). Furthermore, CD146 was shown to be expressed on bone marrow-derived stem cells committed to the smooth muscle lineage (Espagnolle et al., 2014). As shown in Figure 2, smooth muscle cells derived from the bladder are positive for CD146. In summary, CD146+ cells mark a population of smooth muscle-committed cells suitable for use in smooth muscle regeneration and tissue engineering. The method of the present invention allows the isolation of CD146+ iSMCs from skeletal muscle-derived cells.

[0084] 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 fusion-competent desmin+ SMDCs (herein referred to 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. The CD56+ iSMCs obtained according to the present invention may be the most suitable for smooth muscle regeneration.

[0085] Alpha smooth muscle actin (aSMA) is one of the first markers of smooth muscle commitment during development (McHugh, 1995). Its presence is essential for mechanotransduction function and contractility in smooth muscle cells (J. Wang et al., 2006). Therefore, the use of aSMA as a marker for iSMCs aimed at regenerating smooth muscle function is essential. The method of the present invention (Example 2) allows the generation of aSMA+ cells from skeletal muscle-derived cells (Figure 3).

[0086] Desmin is one of the earliest known myogenic markers present in all muscle types. Lack of desmin can lead to muscle degeneration and dysfunction (Capetanaki et al., 1997). Therefore, the use of desmin+ cells for muscle regeneration is preferred. As shown in Figure 3, iSMCs derived from MPCs are desmin positive.

[0087] CD49a or integrin alpha protein (also VLA-1), resulting from 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). The method of the present invention allows for the isolation of CD49a-positive iSMCs from CD49a-negative MPCs or MSCs. As shown in Figure 2, human bladder-derived smooth muscle cells (hBd-SMCs) are also positive for CD49a.

[0088] The function of smooth muscle tissue depends on the presence of highly differentiated smooth muscle cells expressing contractile proteins such as smoothelin (Niessen et al., 2005). Moreover, smoothelin is a well-known marker of fully differentiated smooth muscle cells and is the first marker to disappear when smooth muscle is damaged (van Eys et al., 2007). Therefore, the use of Smoothelin+ cells in the regeneration of smooth muscle tissue may be preferable. With the current method (Examples 2 and 5), we are able to isolate Smoothelin+ iSMCs from Smoothelin- MPCs (Figure 3).

[0089] As mentioned above, the mentioned markers CD146, CD56, aSMA, CD34, desmin, CD49a, smoothelin are important for the identification and / or function of smooth muscle cells, and a combination of markers may be advantageous for identifying iSMCs suitable for smooth muscle regeneration. The method demonstrated herein allows the isolation of iSMCs with the combination of markers treated (Example 2).

[0090] In particular, the present invention provides a method for obtaining iSMCs from skeletal muscle-derived MPCs. The iSMCs obtained from MPCs are CD56+, aSMA+, CD49a+, desmin+, CD146+, and CD34-. The latter markers mentioned above are advantageous for identifying smooth muscle cells, for example, because hBd-SMCs are positive for CD146 and CD49a (Figure 2), and thus are preferred for cells used as smooth muscle regenerating cells. The combination of expression of CD56+, aSMA+, CD49a+, desmin+, CD146+, and CD34- markers is advantageous and novel for iSMCs generated in vitro from human skeletal muscle-derived MPCs.

[0091] Furthermore, the present invention provides a method for obtaining iSMC from skeletal muscle-derived MSC.iSMC derived from skeletal muscle MSC is aSMA+, CD146+, CD49a+, CD56-, and preferably desmin- and / or CD34-.The positive expression of aSMA, CD49a and CD146 and the negative expression of CD56 and preferably 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 more advantageous and novel than previous methods in the art.

[0092] In a further preferred embodiment of the present invention, the method further comprises expanding the skeletal muscle-derived cells after step (a) to preferably obtain 20 to 40 × 10 6 A step (a1) is performed which includes receiving cells.

[0093] In certain preferred embodiments of the invention, skeletal muscle-derived cells are expanded to 50×10 6 Receive cells.

[0094] In a further preferred embodiment of the invention, step (b) is carried out for a period of between 1 and 6 days. In a particularly preferred embodiment of the invention, step (b) is carried out for a period of between 3 and 6 days.

[0095] A further subject of the present invention relates to induced smooth muscle cells (iSMCs) obtained by the method according to the invention.

[0096] In a further preferred embodiment of the invention, induced smooth muscle cells (iSMCs) obtained from MPCs are characterized by positive expression of aSMA, CD49a, desmin, CD56, and CD146, and negative expression of CD34.

[0097] As already demonstrated above, the combination of markers aSMA, CD49a, desmin, CD56 and CD146, or the absence of CD34, in iSMCs from MPCs is advantageous as it phenocopies the natural expression profile of smooth muscle cells by the methods described herein.

[0098] In a further preferred embodiment of the invention, the induced smooth muscle cells (iSMCs) derived from MPCs are non-fusion competent.

[0099] Fusion of mononuclear myogenic progenitor cells (MPCs) or other fusion-competent muscle-derived cells (such as myoblasts) into multinucleated myotubes is a prerequisite for the formation and regeneration of skeletal muscle (Rochlin et al., 2010). However, since smooth muscle tissue in vivo does not consist of multinucleated myotubes but rather of differentiated mononucleated smooth muscle cells, non-fusion competent cells are advantageous for smooth muscle regeneration. Thus, the present invention foresees that iSMCs from MPCs as well as iSMCs from MSCs are non-fusion competent. While MPCs obtained as shown in Example 1 of the present invention are fusion competent, iSMCs obtained as shown in Example 2 of the present invention and ultimately intended for application in smooth muscle tissue are non-fusion competent. This applies to both MPC-iSMCs and MSC-iSMCs according to the present invention, as shown, for example, in FIG. 15.

[0100] In a further preferred embodiment of the invention, induced smooth muscle cells (iSMCs) obtained from MSCs are characterized by positive expression of aSMA, CD49a, and CD146, and negative expression of CD56.

[0101] Preferably, the iSMCs derived from the MSCs are further characterized by negative expression of desmin and / or CD34.

[0102] Skeletal muscle-derived MSCs differ from MPCs in terms of CD56, so MSC-derived iSMCs are also CD56-negative. However, MSC-derived iSMCs are aSMA+, CD146+, and CD34-, which is representative of the smooth muscle commitment of MSC-derived iSMCs, consistent with MPC-derived iSMCs, and is therefore advantageous. Furthermore, the expression of positive mesenchymal markers CD90, CD105, and CD73 in MSC-derived iSMCs according to the present invention is favorable for the mesenchymal nature of the required smooth muscle tissue.

[0103] The expression of the various markers as above is preferably tested in vitro.Furthermore, the expression of the various markers 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 defined above corresponds to their respective in vivo expression.

[0104] Preferably, the induced smooth muscle cells (iSMCs) express functional calcium and / or potassium channels.

[0105] The functionality of smooth muscle tissue depends on the presence of functional voltage-gated calcium and potassium channels, which allow the induction of regulated cell contraction and regulated membrane potential, respectively (Sanders, 2008). Upon neurological stimulation, smooth muscle cell membranes depolarize and open voltage-sensing calcium channels to allow calcium ions to enter the cell from the intercellular space (Sanders, 2008). Following this event triggers a signaling cascade that ultimately results in actin / myosin-induced contraction of smooth muscle cells, which is specifically required for the function of, for example, the internal anal sphincter to contract and retain liquids, gases, and solids from involuntary release from the rectum (Webb, 2003). Furthermore, voltage-gated potassium channels open after neuron-induced depolarization of the smooth muscle membrane, repolarizing the membrane and allowing further depolarization when tracking neuronal signals. Thus, the presence of calcium and potassium channels on iSMCs is suitable for the identification of functional iSMCs in vitro. Functional iSMCs are indeed necessary, for example, to regenerate dysfunctional internal anal sphincters that are poorly functioning in patients with fecal incontinence. The present invention allows the generation of iSMCs from skeletal muscle-derived cells that have both functional voltage-gated potassium and calcium channels, which may be advantageous for their use in smooth muscle regeneration.

[0106] Preferably, the induced smooth muscle cells (iSMCs) are contractile in vitro. One of the typical functions of smooth muscle tissue is contraction (Webb, 2003). To test contractility in vitro, the cells are seeded in collagen gels and the decrease in size of the collagen gel over time is quantified as a measure of contractility. The inventors have found that iSMCs from MPCs and iSMCs from MSCs are contractile compared to the MPCs and MSCs from which they originate.

[0107] Preferably, the induced smooth muscle cells (iSMCs), in particular MPC-iSMCs, obtained according to the method of the invention, preferably in step (b), are CD49e-. The expression of CD49e is preferably tested in vitro. CD49e, also known as integrin alpha 5, is a cell adhesion molecule and forms a heterodimeric receptor with integrin beta 1 that binds to fibronectin, fibrinogen, and fibrillin-1. Fibronectin signaling supported through CD49e may prevent smooth muscle expression, since fibronectin inhibitors were sufficient to increase smooth muscle gene expression. The inventors discovered that iSMCs from mouse MPCs are deficient in CD49e. The deficiency of CD49e may help to reduce fibronectin signaling, and therefore CD49e-iSMCs obtained according to the invention may be more advantageous than cells known in the art in their use for smooth muscle regeneration.

[0108] In an alternative preferred embodiment, the induced smooth muscle cells (iSMCs), in particular MPC-iSMCs, obtained according to the method of the invention, preferably in step (b), are CD49e+. Preferably, the expression of CD49e is tested in vitro.

[0109] Preferably, induced smooth muscle cells (iSMCs), in particular MPC-iSMCs, obtained according to the method of the invention, preferably in step (b), are AChE-. The expression of AChE is preferably tested in vitro. One typical function of skeletal myogenic cells is the expression of active AChE enzyme during in vitro fusion, as it is necessary for the termination of nerve signals at the motor endplate (Thurner et al., 2018). However, since smooth muscles are not innervated by motor neurons, their contraction is not mainly regulated by acetylcholine and requires AChE for termination. We analyzed iSMCs isolated according to Example 3 for AChE activity according to Example 17 and found that iSMCs are AChE- (Figure 14).

[0110] Preferably, the induced smooth muscle cells (iSMCs), in particular MPC-iSMCs, obtained according to the method of the invention, preferably in step (b), are CK-. The expression of CK is preferably tested in vitro. One of the typical functions of skeletal myogenic cells is the expression of active CK enzyme during in vitro fusion, since it is necessary for skeletal muscle contraction (Thurner et al., 2018). However, smooth muscle contraction is not required for smooth muscle cells, since it is not controlled by creatine kinase (CK). The inventors found that iSMCs (Example 3) analyzed for CK activity (Example 17) were CK- (Figure 14).

[0111] In a preferred embodiment, the induced smooth muscle cells (iSMCs), in particular MPC-iSMCs, obtained according to the method of the invention, preferably in step (b), are Pax-7 negative, in particular when tested in vitro for expression of Pax-7. In a further preferred embodiment, the induced smooth muscle cells (iSMCs), in particular MPC-iSMCs, obtained according to the method of the invention, preferably in step (b), are Pax-7 positive, in particular when tested in vitro for expression of Pax-7. 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 muscularis layer of mice leads to a reduction in skeletal muscle and an increase in smooth muscle mass (Woerl et al., 2009).

[0112] In a preferred embodiment, the induced smooth muscle cells (iSMCs), particularly MPC-iSMCs, obtained according to the method of the present invention, preferably in step (b), are SSEA4 negative. In a further preferred embodiment, the induced smooth muscle cells (iSMCs), particularly MPC-iSMCs, obtained according to the method of the present invention, preferably in step (b), are SSEA4 positive. The expression of SSEA4 is preferably tested in vitro. SSEA4 is a cell surface marker found on pluripotent stem cells, known for their extensive proliferation potential, and therefore the risk of tumor formation.

[0113] [Therapy based on induced smooth muscle cells] A further subject of the present invention relates to induced smooth muscle cells (iSMCs) for use in a method for treating a disease or disorder in 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 for treating the human or animal body by surgery or therapy. More particularly, the present invention provides induced smooth muscle cells (iSMCs) for use in cell therapy, in particular smooth muscle cell therapy.

[0114] In a further preferred embodiment of the invention, the disease or disorder is a smooth muscle deficiency. Preferably, the smooth muscle deficiency is selected from the group consisting of anal incontinence, urinary incontinence, reflux disease, gastroparesis, overactive and underactive bladder.

[0115] In a particularly preferred embodiment of the present invention, the disease or disorder is fecal incontinence, particularly passive fecal incontinence.Therefore, the present invention also refers to iSMC for use in the method of treating anal incontinence, urinary incontinence, reflux disease, gastroparesis, overactive and underactive bladder, particularly fecal incontinence, more particularly passive fecal incontinence.

[0116] Preferably, iSMC is injected into the smooth muscle tissue of the subject who needs iSMC.Preferably, iSMC is injected in an amount effective for treating smooth muscle deficiency.The effective amount of compound to be administered can be easily determined by those skilled in the art during preclinical and clinical trials by methods well known to physicians and clinicians.

[0117] Regeneration of necessary smooth muscle tissues, such as weakened, atrophied, or damaged smooth muscle, e.g., sphincters such as the internal anal, internal urethral, ​​lower or upper esophageal sphincter, by cell administration requires that cells are administered locally to the tissue in need. Local administration of iSMCs may regenerate smooth muscle by engraftment of the injected iSMCs at the administration site. Prior art (Chancellor et al., 2001) discloses skeletal muscle-derived cells characterized as desmin+, CD34+, and Blc-2+, and their use in augmenting soft tissues (e.g., smooth muscle), such as the bladder or anal sphincter. In contrast, the present invention discloses the isolation of CD34-, CD56+, desmin+ MPCs, or multipotent CD34-, CD56-, CD73+, and CD105+ MSCs skeletal muscle-derived cells in a first step. From said cells of skeletal muscle, iSMCs can be isolated in a second step following treatment with TGFb1 and heparin. These iSMCs thereby acquire a smooth muscle phenotype, for example by expression of smooth muscle markers such as aSMA and CD146, which may be advantageous for smooth muscle regeneration. Preferably, these iSMCs are used in a method for treating a subject in need thereof by injecting said subject. The inventors have found that iSMCs obtained by the method of the invention are administered to the smooth muscle tissue of the pyloric sphincter, engraft at the injection site and integrate into the smooth muscle tissue.

[0118] Preferably, the iSMCs are administered to the soft tissue in need thereof by multiple injections.

[0119] Multiple injections of SMDCs into skeletal muscle have been shown to improve cell engraftment into muscle (Skuk et al., 2014). The present invention envisions injection of iSMCs into soft tissue, e.g., smooth muscle, at multiple sites in the same tissue continuum. In particular, cells may be administered using multiple needles, each injecting a defined number of cells.

[0120] The iSMCs according to the present invention can be administered in the form of a pharmaceutical composition comprising the iSMCs according to the present invention and a pharma- ceutically acceptable diluent, excipient or carrier. Thus, the present invention also refers to a pharmaceutical composition comprising the iSMCs according to the present invention and a pharma- ceutically acceptable diluent, excipient or carrier.

[0121] In another embodiment of the invention, iSMCs are used in the manufacture of a medicament for the treatment of anal incontinence, urinary incontinence, reflux disease, gastroparesis, overactive and underactive bladder, particularly fecal incontinence, more particularly passive fecal incontinence.

[0122] [Tissue engineering] In a further subject matter of the invention, the induced smooth muscle derived cells are for use in tissue engineering.

[0123] Smooth muscle tissues often form ring / tube-like structures inside the human body, such as blood vessels and the internal anal sphincter. The use of iSMCs obtained herein to generate ring / tube-shaped structures can be used for in vitro engineering of blood vessels or sphincters. In vitro engineered smooth muscle structures may be useful for replacing dysfunctional smooth muscle structures, such as smooth muscle sphincters, for example, in patients with fecal incontinence. The inventors have found that iSMCs obtained by the present invention can reconstruct ring-shaped three-dimensional structures similar to, for example, the internal anal sphincter. The cells in these tissue rings were found to express smooth muscle marker proteins, such as aSMA and desmin. The expression of contractile proteins aSMA and desmin in the tissue rings obtained by the cells of the present invention is necessary for the functionality of the tissue rings, and is therefore advantageous for the use of said tissue rings in tissue replacement.

[0124] Moreover, it has been found herein that iSMCs derived from in vitro engineered sphincters have ultrastructural properties of native smooth muscle, such as highly abundant actin structures and dense bodies, necessary for mechanotransduction and thus functionality of smooth muscle constructs. Furthermore, the tissue engineered sphincters obtained according to the present invention allow the formation of caveolae within the cells. Caveolae are known to be necessary for calcium handling, for example, by excitation-contraction, excitation-transcription and pharmacodynamic coupling (Popescu et al., 2006). Thus, the iSMCs obtained according to the present invention are promising for use in tissue engineering.

[0125] [Drug Screening] In a further subject matter of the invention, the iSMCs are for use in drug screening.

[0126] Using iSMCs to generate smooth muscle structures in vitro may be useful for testing new drugs and their effects on smooth muscle cells in vitro before using potentially harmful drug candidates in animal or human studies.

[0127] [Skeletal muscle-derived cells for the generation of induced smooth muscle cells] A further subject of the invention relates to the use of skeletal muscle-derived cells to obtain induced smooth muscle cells (iSMCs).

[0128] Frequently used state-of-the-art methods use induced pluripotent stem cells (Dash et al., 2016), adipose-derived pluripotent mesenchymal stem cells (G. Wang et al., 2015) or bone marrow-derived pluripotent mesenchymal stem cells (Espagnolle et al., 2014) to obtain smooth muscle cells in vitro, which are of clearly different origin from the skeletal muscle-derived cells used as a source for obtaining induced smooth muscle cells in the present invention. A disadvantage of current state-of-the-art cells, e.g. iPSCs used to obtain smooth muscle cells, is the risk of malignant transformation due to the genetic engineering required to obtain iPSCs. Another state-of-the-art method describes CD34+ skeletal muscle-derived cells as a source of smooth muscle cells. Thereby, said cells are cultured for 2 to 4 weeks in differentiation medium (Lu et al., 2011). For the first time in the present invention, CD34- skeletal muscle-derived cells isolated according to the method of the present invention are used to obtain iSMCs. Since the method of the present invention takes less than one week, the skeletal muscle-derived cells are well suited for isolating iSMCs.

[0129] In a preferred embodiment according to the invention, the skeletal muscle derived cells are oligopotent myogenic progenitor cells (MPCs) characterized by positive expression of CD56, desmin, and negative expression of CD34, or the skeletal muscle derived cells are multipotent mesenchymal stromal cells (MSCs) characterized by positive expression of CD105, CD73, and negative expression of CD34 and CD56.

[0130] The following examples illustrate the invention but are not to be considered as limiting. EXAMPLES

[0131] Example 1 - Isolation of skeletal muscle derived cells (SMDCs) Depending on the isolation method followed, SMDCs were enriched for either human myogenic progenitor cells (MPCs), murine myogenic progenitor cells (mMPCs), or human multipotent mesenchymal stem cells (MSCs).

[0132] [Isolation of myogenic progenitor cells (MPCs) from human skeletal muscle] In detail, skeletal muscle biopsies were taken from the pectoralis major or triceps brachii muscles of incontinent patients. To perform the biopsy, the skin was first opened by an incision of about 1 cm length above the muscle until the fascia of the pectoralis major was reached. After opening the fascia, the skin was opened by an incision of about 1 cm length above the muscle. 3 100 pieces of muscle tissue (biopsies) were taken. The biopsies were transferred directly into biopsy transport medium, which consisted of Ham's F10 basal medium pre-cooled to approximately 4 °C and supplemented with gentamicin (1–5 μg / ml final concentration). The biopsies were stored in biopsy transport medium at 1–11 °C for approximately 26 h. The biopsies were then transferred to a Petri dish filled with 1x PBS. The muscle tissue was separated from the connective tissue using sterile forceps and a scalpel. The muscle tissue was then transferred to another Petri dish filled with 1x PBS and cut into 2–3 mm pieces using a scalpel. 2 After an additional transfer step as above, the tissue pieces were further cut into 1 mm fragments. Finally, the fragments were transferred to a centrifuge tube filled with 1x PBS and centrifuged at 1300 rpm for 10 min. After centrifugation, the supernatant was removed and the muscle tissue was resuspended in 1x PBS supplemented with 8 μg / ml gentamicin. The muscle tissue suspension was then cooled to 2–8 °C for 48 h. After cooling, the muscle tissue suspension was centrifuged at 1300 rpm for 10 min, then the supernatant was removed and resuspended in 2.5 ml of digestion solution containing 1–5 mg / ml collagenase, 2–4% v / v Hepes buffer, 0.1–10% v / v fetal bovine serum and 5–10 μg / ml gentamicin in Ham's F10. The muscle tissue suspension was then incubated at 37 °C, 5% CO2 for 6 to 20 h. The suspension was then centrifuged at 1300 rpm for 10 min, the supernatant was removed, and the pellet was resuspended in medium containing 10–20% v / v FCS, 1–3 ng / ml bFGF, and 3–10 μg / ml gentamicin in Ham's F10 and seeded into cell culture flasks. SMDCs attached to the bottom of the culture flask were further maintained by changing the medium every 3–4 days and by detaching and subculturing after reaching confluence. 1 × 10 7 From 5×10 7 The cells were subcultured until the SMDC reached 1.

[0133] [Isolation of mouse skeletal muscle-derived myogenic progenitor cells (mMPCs)] Mouse MPCs were obtained from skeletal muscle biopsies of Gt(ROSA)26Sortm4(ACTB- tdTomato,-EGFP)Luo / J, or in other words TdTomato, mice (Jackson Laboratory, Maine, USA). Adult mice were sacrificed by cervical dislocation, followed by pinching the skin on the back to remove it. Skeletal muscles were then obtained from the longissimus dorsi, gastrocnemius, and tibialis anterior muscles using scissors and a scalpel. The muscles were transferred to a sterile Petri dish and covered with 1x PBS. Remaining connective tissue was then removed from the skeletal muscles using tweezers and a scalpel and discarded. Muscle tissue was then digested using a skeletal muscle dissociation kit (MiltenyiBiotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. To separate myogenic precursor cells (mMPCs) from non-myogenic SMDCs, a satellite cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the manufacturer's instructions. Collected mMPCs and non-myogenic SMDCs were centrifuged as above and resuspended in mouse growth medium consisting of DMEM / Ham's F12 supplemented with 20% FCS and bFGF. Mouse SMDCs were cultured in collagen-coated culture flasks, prepared by covering the surface of the culture flask with collagen I from rat tails diluted 1:10 in 1x PBS, for 1 h at 37 °C. Subculture was performed as for human SMDCs. Finally, the cells were detached from the walls of the cell culture vessel, resuspended in medium, and used immediately or frozen and stored in liquid nitrogen until further use.

[0134] [Isolation of multipotent mesenchymal stem cells (MSCs) from human skeletal muscle] MSCs were isolated according to Thurner et al. 2018. First, SMDCs were isolated from muscle biopsies (pectoralis major or latissimus dorsi) and expanded under cGMP environment. Cells were maintained by standard cell culture methods. Briefly, cells were cultured in growth medium containing Ham's F-10 basal medium supplemented with 10% FCS (inactivated at 57°C for 40 min), bFGF and gentamicin and incubated at 37°C and 5% CO2. Growth medium was changed every 2 to 3 days. For subculture and harvesting, cells were washed once with 1x PBS and incubated with 1x trypsin solution at 37°C for 5 min. Cells were rinsed with growth medium, centrifuged at 400×g for 10 min, the supernatant was discarded and the pellet was resuspended in growth medium. MSCs were then purified by magnetic activated cell sorting (MACS). Therefore, the human CD56 MicroBeads kit (MiltenyiBiotec GmbH, Bergisch Gladbach, Germany) was used. Briefly, after harvesting and counting, cells were centrifuged at 400 × g for 10 min, the supernatant was discarded, and the cells were resuspended in 10 mL of MACS buffer. After another centrifugation step (400 × g for 10 min), the pellet was resuspended in 80 μL of MACS buffer. Subsequently, 1 × 10 7 Magnetic CD56 antibody (20 μL per cell) was added and incubated for 15 min at 4 °C. Cells were then sorted using a Mini MACS separator and CD56-MSCs were collected in the flow-through. Finally, cells were resuspended in culture medium and frozen in liquid nitrogen until further use or immediate use.

[0135] Example 2 - Transdifferentiation of SMDCs into iSMCs and their isolation MPC, mouse MPC or MSC obtained in Example 1 were seeded on the wall of the culture vessel and cultured in growth medium until about 70% confluence. Next, the cells were washed once with DMEM / F12 (Thermo Scientific, MA, USA). Next, the cells were covered with smooth muscle differentiation medium consisting of DMEM / F12 supplemented with recombinant human TGFb1 (Thermo Scientific, MA, USA), heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich Co. LLC, MO, USA), heat-inactivated (57°C, 40 min) 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, the cells were cultured in smooth muscle differentiation medium at 37°C and 5% CO2 for 3-6 days. The medium was changed every 3-4 days. For isolation, cells were detached from the walls of the culture vessel and collected in suspension.

[0136] [Example 3 - Differentiation potential of SMDC] For in vitro adipogenic, chondrogenic, and osteogenic differentiation, 500,000 cells obtained by Example 1 were seeded in a 6-well plate (NUNC, Thermo Scientific, MA, USA) and cultured in growth medium at 37°C and 5% CO2 for 24 hours. Then, the cells were washed once with 5ml DMEM / Ham's F12 and covered with 5ml of adipogenic, chondrogenic, or osteogenic differentiation medium, respectively. Adipogenic, chondrogenic, and osteogenic differentiation medium consisted of StemXVivo™ Osteogenic / Adipogenic Base Medium (R&D Systems Inc., MN, USA), StemXVivo Human / Mouse / Rat Adipogenic (R&D Systems Inc., MN, USA), StemXVivo Human Osteogenic Supplement (R&D Systems Inc., MN, USA), or STEMPro® Chondrogenic Supplement (Gibco®, Thermo Scientific, MA, USA), respectively, according to the manufacturer's instructions. Gentamicin (Sandoz GmbH, Austria) was further added to the differentiation medium to reach a final concentration of 3.83 μg / ml. Cells were cultured in the respective differentiation medium for 14 days each, with the medium being changed every 2–3 days. After 14 days of culture, successful differentiation was assessed by the presence of adipocytes, chondrocytes and osteocytes visualized by staining with Oil Red O (adipocytes), Alcian Blue (chondrocytes) and Alizarin Red S (osteocytes), respectively. Quantification of Oil Red O, Alcian Blue and Alizarin Red S staining was performed in microscopic images of several individual experiments by the Image J software package. For this, images were loaded and the color channels were split. The red channel was used for Oil Red O and Alizarin Red S staining, and the blue channel for Alcian Blue staining. The background was removed by setting a common threshold, and the average pixel intensity per field was obtained for quantification by Image J. Statistical comparisons were performed by unpaired t-test, with p<0.05 considered as significant (*). p<0.01, p<0.001 were visualized as * or **, respectively.In cell cultures in 24-well Nunclon™ Delta Surface plastic plates (Thermo Scientific, MA, USA), skeletal muscle differentiation was initiated by replacing the growth medium with skeletal muscle cell differentiation medium (500 mL, PromoCell 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) as previously described ( Thurner et al., 2018 ).

[0137] MPCs and MSCs obtained according to Example 1 were tested for in vitro differentiation into adipogenic, chondrogenic, osteogenic and skeletal myogenic lineages under appropriate culture conditions. Staining of adipocytes, chondrocytes, osteocytes and myotubes was performed as described above. No oil red o and alizarin red s positive cells were present in MPCs and only low levels of alcian blue cells could be detected. In MSCs, oil red o, alcian blue and alizarin red s positive cells were found after culture in adipogenic, chondrogenic and osteogenic differentiation media, respectively (Figure 1A, B), confirming enrichment of multipotent cells and multipotent mesenchymal stem cell status as defined by the International Society for Cell Therapy (Dominici et al., 2006). Desmin positive multinucleated myotubes were detected only in MPCs (Figure 1A). Quantification of Oil Red O, Alcian Blue and Alizarin Red S staining intensity after in vitro adipogenic, chondrogenic and osteogenic differentiation, respectively, as well as fusion index calculation after skeletal myogenic differentiation of MSCs and MPCs revealed significantly higher staining intensity in Oil Red O (p=0.0117), Alcian Blue (p=0.0020) and Alizarin Red S (p=0.0012) staining of MSCs compared to MSCs, whereas a significantly higher fusion index (p=0.0007) was found within MPCs compared to MSCs (Figure 1B). Thus, MPCs are mesenchymal oligopotent cells and committed to the myogenic lineage. Moreover, MSCs are multipotent and capable of adipogenic, chondrogenic and osteogenic differentiation in vitro.

[0138] Example 4 - Surface marker expression To measure the expression of surface markers, flow cytometry was performed on a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, cells obtained according to Example 1 were harvested by overlaying with 1x trypsin for 5 min at 37°C, centrifuged at 400xg and resuspended in 1x PBS supplemented with 1% FCS. 40000 cells were resuspended in 195μl 1x PBS and incubated in 1.5mL Eppendorf tubes at 4°C in the dark for 30 min after addition of 5μl 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). Cells were then washed with 1 mL PBS, centrifuged at 400×g for 10 min, and resuspended in 195 μL of 1×PBS in a 96-well round-bottom plate. 5 μL of the viability dye 7-aminoactinomycin D (Beckman Coulter Inc., France) was then added to each reaction, and the plate was incubated for 10 min in the dark at room temperature. Finally, cell events were acquired using Guava InCyte™ v.2.3 software. Histograms and dot plots were generated with a sample flow rate of 1.8 μL / mL and a minimum of 5000 events. Positive staining was obtained by comparison to isotype controls set as at least 95% negative, or by comparison to control (negative) cells.

[0139] To characterize MPC and MSC, at least four samples from individual patients obtained by the method used in Example 1, and iSMC derived therefrom by the method used in Example 2, were tested for the presence of mesenchymal (CD105, CD90, and CD73), hematopoietic (CD34), myogenic (CD56), and smooth muscle lineage (CD146) markers. A mean % positive cells ≥ 50 was considered positive, whereas a mean % positive cells < 50 was considered negative. All cell types were thereby found to be positive for CD105, CD90, and CD73, but negative for CD34 (Figure 2). Furthermore, MPC and iSMC derived therefrom were CD56+, whereas MSC and iSMC derived therefrom were CD56-.

[0140] Interestingly, expression of surface markers CD146, associated with 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 the derived iSMCs ( Figure 2 ).

[0141] Example 5 - Intracellular marker expression Immunofluorescence staining was performed to detect the expression of intracellular markers directly on gelatin-covered 24-well plates or on glass coverslips placed in 6-well plates, as previously described (Thurner et al., 2018). For fluorescent immunolabeling of alpha smooth muscle actin (aSMA), smoothelin, or desmin, cells were incubated with mouse anti-actin alpha smooth muscle (Sigma-Aldrich Co. LLC, MO, USA), mouse anti-smooth muscle (Merck Millipore, MA, USA), anti-smooth muscle myosin heavy chain (Merck Millipore, MA, USA), or rabbit anti-desmin (Thermo Scientific, MA, USA) antibodies, respectively, diluted 1:100 in blocking medium. Secondary goat anti-mouse Alexa488 or donkey anti-rabbit Alexa547 conjugated antibodies (Thermo Scientific, MA, USA) diluted 1:200 in blocking medium were used. Nuclear counterstaining was performed by incubating cells with Hoechst33342 (Sigma-Aldrich Co. LLC, MO, USA) diluted to a final concentration of 2 μg / mL in PBST (0.1% Triton X-100). Cells were mounted with Entellan® (Merck Millipore, MA, USA) and sealed with glass coverslips. Staining was compared to (1) a procedure without primary antibody and (2) cells negative for the tested antibodies. 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 antibody staining was divided by the total number of cells (nuclei) assessed by Hoechst staining. Mean and standard error values ​​were calculated to compare cells obtained by Examples 1 and 2.

[0142] MPC and MSC obtained according to Example 1, and iSMC derived from them as described in Example 2, were analyzed for expression of intracellular contractile smooth muscle protein (aSMA, smoothelin) as well as the general myogenic marker desmin by fluorescent immunostaining. MPC and MSC were aSMA- and smoothelin-. In contrast, iSMC isolated from both MSC and MPC were found to be aSMA+ (Figure 3). Furthermore, iSMC isolated from MPC in Example 2 were found to be smoothelin+. Analysis of intracellular desmin expression revealed that MSC and iSMC isolated therefrom were desmin-. In contrast, both MPC and iSMC isolated therefrom were desmin+ (Figure 3).

[0143] Example 6 - Gene Expression 1×10 MPCs or MSCs obtained as shown in Example 1 and iSMCs derived therefrom as shown in Example 2 6 Total RNA was isolated by RNEasy kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Sample preparation for microarray hybridization was performed as described in the NuGEN Ovation PicoSL WTA System V2 and UGENEncore Biotin Module manuals (NuGEN Technologies, Inc, San Carlos, CA, USA). Hybridized arrays were washed and stained with an Affymetrix Fluidics Station FS450, and fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scanning functions were controlled by Affymetrix GeneChip Command Consolev 4.1.3 software. Sample processing was performed at the Affymetrix service provider and core facility "KFB - Center of Excellence for Fluorescent Bioanalytics" (Regensburg, Germany).

[0144] The summarized probeset signals on the log2 scale were calculated using the RMA algorithm in Affymetrix GeneChip Expression Console v1.4, and the probeset IDs with the highest log2 fold change between MPCs and MPC-iSMCs were used for subsequent analysis and comparison with the log2 fold change between MSCs and MSC-iSMCs. An overview of all analyzed genes and their respective log2 fold changes between MSCs and MSC-iSMCs, and between MPCs and MPC-iSMCs, are shown in (Figure 12). Log2 fold changes ≥ 1 were considered to indicate upregulation and are therefore marked with an asterisk (*). To visualize the log2 fold changes and to perform hierarchical clustering and k-means clustering according to Euclidean distance, heatmaps were generated using Multiple Expression Viewer (MeV 3.1.0) software.

[0145] To study the changes in gene expression associated with the isolation of iSMCs in the present invention, microarray analysis was performed. Smoothellin (SMTN), calponin 1 (CNN1), tropomyosin 1 (TPM1), transgelin (TGLN, SM22), integrin-alpha-3 (ITGA3), integrin-alpha-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 commitment gene desmin (DES) (Capetanaki et al., 1997), and all genes required for vascular smooth muscle contraction (KEGG PATHWAY: vascular smooth muscle contraction-Homo sapiens (human), not detected) were analyzed in detail. The changes in gene expression between MPCs and derived iSMCs were compared with those between MSCs and derived iSMCs. Considering a log2 FC ≥1 and a log2 FC ≤-1 as up- and down-regulation, respectively, 20.33 percent of the 123 genes tested were up-regulated and only 3.25 percent were down-regulated during differentiation of MPCs to MPC-iSMCs, suggesting differentiation towards a smooth muscle cell phenotype. Upregulated genes of KEGG clusters or known smooth muscle marker genes in MPC-iSMCs compared to MPCs were PPP1R14A, KCNMB1, PLCB4, ACTG2, ITPR1, ADCY6, CALCRL, KCNMA1, GNA13, CNN1, ADCY2, KCNMB4, GUCY1A3, ARAF, ITGA1, PPP1R12A, MAPK1, CALD1, KCNMB2, PRKACB, ARHGEF11, PPP1R12C, ITPR2, PLCB1, and SMTN ( Figure 12 ).

[0146] Considering log2 FC ≥ 1 and log2 FC ≤ -1 as up- and down-regulation, respectively, 12.20% of the 123 genes tested were up-regulated and only 3.25% were down-regulated during differentiation of MSCs to MSC-iSMCs, suggesting differentiation towards a smooth muscle cell phenotype. Up-regulated genes of KEGG clusters or known smooth muscle marker genes in MSC-iSMCs compared to MSCs were ACTA2, ACTG2, CALD1, GNAQ, ITPR1, MAPK1, MYL9, KCNMA1, PLCB4, PPP1R14A, PRKCE, CNN1, TPM1, TAGLN, and ITGA1 (Figure 12). The finding that gene expression of ITGA1, which encodes CD49a, and SMTN, which encodes smoothelin protein, were found to be upregulated in MPC-iSMCs supports our finding that the percentage of CD49a- and smoothelin-positive cells was increased in MPC-iSMCs compared to MPCs, thus confirming smooth muscle marker expression in MPC-iSMCs.The finding that gene expression of ITGA1, which encodes CD49a, was found to be upregulated in MSC-iSMCs supports our finding that the percentage of CD49a-positive cells was increased in MSC-iSMCs compared to MSCs, thus confirming smooth muscle marker expression in MPCs-iSMCs.

[0147] Although CD146 (MCAM) surface protein expression was upregulated in MPC-iSMCs, MCAM gene expression was not found to be upregulated in microarray experiments, suggesting post-transcriptional regulation of CD146 expression. Furthermore, k-means cluster analysis of log2 FC in which gene expression was altered in MPCs and MSCs during differentiation into MPC-iSMCs and MSC-iSMCs led to the identification of genes that were similarly upregulated (Figure 4A) and downregulated (Figure 4B) between MPCs and MSCs when their iSMCs were isolated, respectively. PP1R14A, ACTG2, PLCB4, ITPR1, MAPK1, CNN1, ITGA1, and KCNMA1 were upregulated in both MPCs and MSCs, whereas PLA2G2A was downregulated in both cell types. Overall, 75.61 percent of the genes tested were similarly upregulated, downregulated, or neither in MPCs and MSCs upon differentiation into iSMCs. Although more upregulated genes and fewer downregulated genes were found in MPCs (20.33% upregulated and 3.25% downregulated) than in MSCs (12.20% upregulated and 5.69% downregulated), no significant differences were found in the percentages of upregulated and downregulated genes between MPCs and MSCs (Figure 4C).

[0148] [Example 7 - Fusion Ability] The fusion ability of MPCs and iSMCs according to Examples 1 and 2, respectively, was evaluated according to their fusion index (FI). To determine the fusion index (FI), cells induced for skeletal muscle differentiation in Example 3 were washed twice with PBS and fixed with 4% PFA for 10 min. Then, cells were washed three times with PBS and stained with 2 μg / mL Hoechst33342 solution for 20 min. For each sample, at least three fields were captured during immunofluorescence imaging, and phase contrast images were overlaid to allow easy detection of nuclei and cell boundaries. After calculating the average of all analyzed fields, the fusion index was calculated for each captured field by dividing the number of nuclei in the tube by the total number of nuclei per field. Only cells with at least three nuclei were considered as myotubes. For statistical analysis, at least three populations derived from different patients were analyzed for each group.

[0149] Quantification of FI showed that significantly more MPCs underwent skeletal myogenesis compared to iSMCs isolated from MPCs in Example 2, suggesting that isolating iSMCs from MPCs reduces the skeletal myogenesis potential of MPCs (Figure 5A). Furthermore, tubes formed by iSMCs contained significantly fewer nuclei than tubes formed by MPCs (Figure 5B). Taken together, MPCs appear to be fusion-competent, whereas iSMCs derived from MPCs appear to be non-fusion-competent.

[0150] Example 8 - Electrophysiology Patch clamp analysis was performed on MPCs and iSMCs obtained according to examples 1 and 2, respectively, according to previously published protocols (Park et al., 2013), with minor experiment-specific adaptations. The procedure is as follows: Electrophysiological recordings were performed in the whole-cell configuration using an Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City). Patch pipettes with resistances of 1 to 4 MΩ were made from borosilicate glass (GC150F-7.5, Clark Electromedical Instruments, UK) and filled with pipette solution. All data were digitized using a DIGIDATA 1200 interface (Axon Instruments, Foster City), smoothed by a 4-pole Bessel filter, and stored on disk. Current traces were sampled at 10 kHz and filtered at 2 kHz. The pClamp software package (version 10.0 Axon Instruments, Inc.) was used for data acquisition. Microcal Origin 7.0 was used for analysis. Reagents were obtained from Sigma-Aldrich unless otherwise stated. Inward currents of voltage-gated Cav channels were evoked by applying 500 ms depolarizing pulses from a holding potential of -50 to 50 mV. Superimposed current traces of Kv channels were evoked by step depolarizing pulses between -80 and 60 mV in 20 mV steps from a holding potential of 80 mV in MPCs, MPC-iSMCs, and hBd-SMCs.

[0151] The cells obtained by Example 2 were found to show neither voltage-sensitive inward nor outward currents of voltage-gated calcium or potassium channels, respectively. In contrast, iSMCs derived from MPCs in Example 2 showed both voltage-sensitive inward and outward currents, as also seen in hBd-SMCs (FIG. 6). In summary, isolation of iSMCs by incubation with TGFb1 and heparin leads to functional maturation.

[0152] [Example 9 - Collagen gel lattice contraction] To measure the contractility of MSCs and MPCs obtained according to Example 1, and iSMCs derived from them according to Example 2, they were seeded on collagen gel lattices and the percent gel size reduction was quantified. In detail, the medium was removed from subconfluent cells in a standard cell culture vessel, and the cells were washed twice with 1×PBS. The cells were then covered with trypsin and incubated at 37° C. for 5 min. The cells were then detached by tapping the walls of the culture vessel and resuspended after the addition of DMEM / Ham's F12 basal medium. The cells were then centrifuged at 400×g for 10 min.

[0153] Remove the supernatant and resuspend the cell pellet in DMEM / Ham's F12 at 6 × 10 cells per ml. 5 cells were obtained. For each gel, 400 μl of cell suspension was mixed with 200 μl of 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 a well of a 24-well plate (NUNC, Thermo-Fisher Scientific, MA, USA). The 24-well plate was then incubated at 37 °C for 30 min to allow the formation of gels. After that, each gel was covered with 500 μl of DMEM / Ham's F12 and floated to the surface by releasing it from the bottom of the 24-well plate using a sterile pipette tip. Finally, the gels were incubated at 37 °C and 5% CO2 for 24 h to allow the contraction of the gel by the contained cells. To quantify the contraction of the gels, stereomicroscope pictures were taken and the area of ​​the gel was calculated using FIJI (image J) software.

[0154] MPC and MSC isolated according to step (a) of the present invention (Example 1) were found to be non-contractile in a collagen gel lattice contraction assay. In contrast, iSMC isolated from MPC and MSC according to step (b) of the present invention (Example 2) showed significantly higher contractility than cells derived from (MPC, MSC). In summary, SMDC transdifferentiated into iSMC and isolated according to step (b) (Example 2) were found to be contractile compared to cells isolated in step (a) (Example 1; Figure 7).

[0155] Example 10 - Smooth muscle regeneration using iSMCs To test the potential of iSMCs obtained according to Example 2 for smooth muscle regeneration, iSMCs derived from mouse MPCs (obtained according to Example 1) were injected into the pyloric sphincter of adult female SHO-PrkdcscidHrhr mice. For this, the mice were first anesthetized by intraperitoneally applying 100 mg / kg ketamine, 10 mg / kg xylazine and 3 mg / kg acepromazine. An eye protection cream was applied during the procedure. Cryopreserved cells were freshly thawed, washed once with 1x PBS and centrifuged at 400xg for 10 min before resuspending the cells in 1x PBS to a final concentration of 40 000 000 cells / ml. Meanwhile, the mice receiving the cells were placed on a heating plate to maintain their body temperature at 37°C. Then, 25 μl of cell suspension (containing 1 000 000 cells) was mixed with 5 μl of FluoSpheres® polystyrene beads, 15 μm yellow-green or blue (Thermo-Fisher Scientific, MA, USA), which was necessary to track the location of the injection after surgery. For iSMC injection into the pyloric sphincter, after performing a midline laparotomy, the pyloric sphincter area was located and 30 μl of cell-fluosphere mixture was applied using a 28G needle attached to a 1 ml syringe. The peritoneum and musculocutaneous layers were closed separately by continuous sutures with 6-0 Ethicon PDS and absorbable monofilament. After surgery, 200 mg of Novargin (Metamizol®) per kilogram of body weight was administered subcutaneously for 3 days. 12 weeks after cell injection, mice were sacrificed by cervical dislocation to obtain and image the pyloric sphincter.

[0156] Imaging of freshly isolated pyloric sphincter regions was performed with IVIS Spectrum (PerkinElmer, MA, USA) using Living Image® software version 4.5.2 (PerkinElmer, MA, USA) according to the manufacturer's instructions. Briefly, pyloric sphincter of injected and control SHO mice was placed on a glass Petri dish and placed in the IVIS system. Fluorescent pictures were taken at a height of 2 cm with automatic exposure times of the corresponding absorption and emission wavelengths of TdTomato and yellow Fluosphere beads. To remove background signals, the post-mortem signal intensity was adjusted by comparison with sphincter explants from control mice.

[0157] For histological analysis, animals were deeply anesthetized with isoflurane and sacrificed by cervical dislocation. Tissues of interest were immediately dissected and cryofixed by immersion in liquid nitrogen-cooled 2-methylbutane. Tissues were cut at 15 μm on a Leica 1950 cryostat, and sections were collected on Superfrost plus slides and stored at −20 °C until further processing. For immunohistological analysis, sections were fixed in 4% PFA and washed in PBS containing 0.1% Tween-20 (Sigma-Aldrich). Blocking and antibody dilution 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). Primary antibodies against tdTomato (Sicgen) or aSMA (Thermo Scientific, MA, USA) were diluted 1:100 in blocking medium after overnight incubation at 4°C. 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 LSM 710 confocal microscope and ZEN 2011 Black Software (Carl Zeiss).

[0158] We found that iSMCs isolated according to Example 2 from TdTomato reporter protein expressing MPCs obtained according to Example 1 were detectable and colocalized with the fluorescent beads co-injected at the site of the pyloric sphincter 12 weeks after transplantation, suggesting that the iSMCs had engrafted at the injection site. Histological examination followed by fluorescent immunostaining for TdTomato and aSMA suggested that TdTomato-positive iSMC cells were found within the circular muscle of the pyloric sphincter and the muscularis mucosae near the co-injected fluorescent beads (Figure 8). TdTomato-positive iSMC cells within the smooth muscle layer of the pyloric sphincter also expressed aSMA protein, suggesting that not only was the engraftment into smooth muscle tissue preserved, but also the phenotypic characteristics of smooth muscle cells after engraftment, which are necessary for smooth muscle regeneration (Figure 8).

[0159] Example 11 - Tissue engineered smooth muscle To test the suitability of the cells obtained by the method herein for use in tissue engineering, 3D cell culture was performed. Using cells obtained according to example 1 or 2, a method was adopted that allows the generation of ring-shaped sphincter-like structures already known in the art (Gwyther et al., 2011). To produce a culture vessel for 3D culture, a template was made in stainless steel (inner diameter 4 mm, outer diameter 10 mm) with a ring-shaped engraving for the production of an autoclavable PDMS negative template. A 2% (w / v) agarose solution was then prepared by weighing 5 g of low-gelling agarose (Sigma-Aldrich, MA, USA) in a Schott® flask filled to 250 ml with Ham's F10 basal medium. Both the PDMS template and the agarose solution were then autoclaved for 20 min at 121°C. The PDMS template was then filled with the agarose solution under sterile conditions, followed by incubation at room temperature for 1 h to allow the agarose to solidify. In the next step, the solidified agarose was detached from the PDMS template, and cubes each containing one ring-shaped template were cut out of the agarose, transferred into a 6-well plate filled with 2 ml of Ham's F10 basal medium, and stored at 37°C until further use. For 3D culture, the cells obtained in Example 1 were centrifuged at 400 × g for 10 min, and the cell pellet was resuspended in culture medium to obtain 5 × 10 6The cells were allowed to reach a concentration of 1000 cells / ml. Then, 200 μl of the cell suspension was added to the agarose template and incubated at 37° C. for 48 hours without further disturbance to allow the formation of a ring of cells. After the incubation period, the medium outside the template in the 6-well plate was discarded and the 6-well plate was carefully filled with 5 ml of medium used in Example 1 to generate 3D cultured MPCs or MSCs, or the latter used in Example 2 to generate 3D cultures of iSMCs. The cells were cultured for 6 days and allowed to mature at 37° C. and 5% CO2 until analysis. After 6 days of culture, the cells were analyzed by standard light microscopy, cryosectioning followed by immunostaining (Example 12) or H&E staining, scanning electron microscopy or semi / ultrathin sectioning followed by toluidine blue staining or transmission electron microscopy. MPCs derived according to Example 1, transdifferentiated towards iSMCs as described in Example 2, formed ring-like sphincters by using the method described above based on Gwyther et al., 2014 (Figure 9).

[0160] Example 12 - Frozen sectioning and immunostaining For histological analysis of the ring-shaped 3D cultures (bioengineered sphincters) obtained according to Example 11, the rings were carefully removed from the agarose template using a sterile spoon, washed by transferring to an Eppendorf tube filled with 1x PBS, and then fixed and permeabilized by immersion in MetOH pre-cooled at -20°C for 5 min. After removing half of the solution, the rings were then washed by diluting MetOH three times with 1x PBS. The bioengineered sphincters were cryofixed by immersion in 2-methylbutane cooled with liquid nitrogen. The specimens were cut at 15 μm on a CM1950 cryostat (Leica, Germany) and sections were collected on Superfrost plus slides (Thermo-Fisher Scientific, MA, USA) and stored at -20°C until further processing. Blocking and antibody dilution were performed using a 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). Primary antibody aSMA (Thermo-Fisher Scientific, MA, USA) was diluted 1:100 in blocking medium after overnight incubation at 4°C. Secondary antibody (Thermo-Fisher Scientific, MA, USA) was diluted 1:500 and applied for 4 h 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 min at room temperature. Sections were then mounted using Prolong Gold Antifade (Thermo-Fisher Scientific, MA, USA). Fluorescent images were acquired using a Nikon Eclipse TE2000-U inverted microscope.

[0161] The cells in the tissue ring obtained according to Example 10 were found to be positive for alpha smooth muscle actin and desmin (Figure 10). In the tissue ring derived from MPC, aSMA was expressed by cells especially in the outer layer of the ring, whereas in the tissue ring containing iSMC, aSMA expressing cells were distributed throughout the ring. In particular, the cells in the ring formed from MPC differentiated into iSMC during 3D culture in combination with Examples 2 and 10 were found to express more aSMA than the cells in the ring formed from MPC (Figure 10). In contrast, desmin was expressed throughout the tissue ring in both MPC-derived and iSMC-derived tissue rings (Figure 10).

[0162] Example 13 - Scanning Electron Microscopy Scanning electron microscopy was performed at the Department of Histology and Embryology of the Medical University of Innsbruck, with the grateful help of Angelika Floerl and Kristian Pfaller. For the analysis of the 3D cultured cells obtained according to examples 1 and 2 (described in section 3.2.26) by scanning electron microscopy, rings were released from the agarose templates, washed once in a 1.5 ml Eppendorf® tube containing 1× PBS, then fixed in pre-cooled (−20° C.) MetOH, followed by post-fixation for 1 h in 1% osmium tetroxide, dehydrated in EtOH and critical point dried in a Bal-Tec CPD 030 critical point dryer (Balzers, Lichtenstein). Samples were mounted with conductive carbon cement Leit-C after Goecke (Plano GmbH, Wetzlar, Germany) on aluminum stubs (Balzers) sputter-coated with 15 nm Au / Pd and examined with a Gemini 982 scanning electron microscope (Carl Zeiss). MPCs obtained according to Example 1 and transdifferentiated towards iSMCs in 3D culture (Example 2) as described in Example 2 appeared elongated and appeared as an integral part of the tissue ring on the surface of the tissue ring formed in Example 10 (Figure 9). The cells appeared intact on the surface of the ring and were potentially viable (Figure 9).

[0163] Example 14 - Transmission electron microscopy Transmission electron microscopy was performed on tissue rings obtained according to Example 10. Thus, the rings were released from the agarose template and transferred to a 1.5 ml Eppendorf tube filled with 1x PBS for washing. The samples were then transferred to new Eppendorf tubes filled with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for fixation and storage (4°C). After at least 24 hours of fixation, the rings were washed twice with phosphate buffer and fixed with 1% OsO4 for 45 minutes. The rings were then washed three times with distilled water for 15 minutes each. The rings were then dehydrated every 30 minutes in increasing EtOH concentrations (70, 80, 90, 100%) and incubated twice in fresh 100% acetone for 20 minutes each. The specimens were then embedded in a 2:1 acetone-Epon mixture for 150 minutes, followed by overnight incubation in a 1:2 acetone-Epon mixture. Finally, the rings were incubated for 24 h in pure Epon rotating on a rotator with Epon replaced after 8 h. For polymerization of Epon, the rings were incubated at 60°C for 24 h. Epon-embedded specimens were trimmed using Ultratrim (Reichert) and ultrathin sections were obtained using an Ultracut S (Reichert). Ultrathin sections were observed at 80 kV using a CM120 TEM (Philips / FEI) equipped with a MORADA CCD camera (Olympus / SIS).

[0164] Transmission electron microscopy of tissue rings obtained according to Example 10 revealed that cells formed caveolae close to their plasma membrane (Fig. 11A and B). Furthermore, cells within the tissue rings had predominant actin structures (Fig. C), and some of the cells were found to have formed dense packaging structures such as dense bodies (Fig. D).

[0165] Example 15 - Surface marker expression in mouse MPC-iSMCs To measure surface marker expression of mouse MPC-iSMCs, flow cytometry was performed on a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, mouse MPC-iSMCs obtained according to Example 1 were used for an antibody staining procedure for a commercially available mouse cell surface marker screening panel (BD biosciences, NJ, USA). Thus, cells were cultured in growth medium at 1.25 × 10 per ml. 6 Cells were suspended at a concentration of 100 μl and aliquots of the cell suspension were mixed with all primary antibodies in a 96-well plate according to the manufacturer's instructions. After incubating the mixtures for 30 min at 4°C, 100 μl of growth medium was added to each well, the plates were centrifuged at 300×g for 5 min, the supernatant was removed, 200 μl of growth medium was added to each well, further centrifugation, removal of the supernatant, and resuspension of the cells in 100 μl of biotinylated secondary antibody (prepared according to the manufacturer's instructions). After incubating the cells again for 30 min at 4°C, 100 μl of growth medium was added to each well, the plates were centrifuged at 300×g for 5 min, the supernatant was removed, 200 μl of growth medium was added to each well, further centrifugation, removal of the supernatant, and resuspension of the cells in 100 μl of Alexa647-conjugated streptavidin (prepared according to the manufacturer's instructions). After again incubating the cells for 30 minutes at 4°C, 100 μl of growth medium was added to each well, the plate was centrifuged at 300×g for 5 minutes, the supernatant was removed, 200 μl of growth medium was added to each well, another centrifugation, removal of the supernatant, and resuspension of the cells in 200 μl of Ham's F10 supplemented with 10% FCS was performed. Finally, cell events were acquired using Guava InCyte™ v.2.3 software. Histograms and dot plots were generated with a minimum of 5000 events at a sample flow rate of 1.8 μL / mL. Positive staining was obtained by comparison with isotype controls set as at least 95% negative, or by comparison with control (negative) cells. The mouse iSMCs of Example 3 were found to be CD49e− (Figure 13).

[0166] Example 16 - Analysis of intracellular markers by flow cytometry Briefly, MPC and MPC-iSMC obtained according to Example 1 were collected by covering the adherent cells with 1× trypsin for 5 min at 37° C., and then centrifuging the detached cells at 400×g. Cells were counted and sorted to achieve 50,000 cells / reaction, then centrifuged at 400×g, before being resuspended in BD Cytofix / Cytoperm fixation and permeabilization solution (BD Biosciences, Pharmingen™) and incubated at 4° C. for 20 min. Cells were then washed with BD Perm / Wash buffer (diluted 1:10 in aqua dest) (BD Biosciences, Pharmingen™) and centrifuged. Cells were then resuspended in 1× PBS and incubated with IgG isotype control-Alexa488 (Bioss Antibodies Inc., MA, USA) or anti-Pax-7-Alexa488 (Bioss Antibodies Inc., MA, USA) for 1 h at 4° C. in the dark. Cells were subsequently centrifuged and washed with BD Perm / Wash buffer (diluted 1:10 in aqua dest) and resuspended in 1x PBS after the final centrifugation step. Cell events were acquired using Guava InCyte™ v.2.3 software. Histograms were generated with a minimum of 3000 events at a sample flow rate of 1.8 μl / ml. The percentage of positive cells was obtained by comparison with the isotype control, which was set as 99% negative.

[0167] Example 17 - Enzyme activity analysis [Acetylcholinesterase activity measurement] <Reagents and standard preparation> American Public Health Association (APHA) phosphate buffer, pH 7.2 (Sigma-Aldrich Co. LLC, Germany) was prepared according to the manufacturer's instructions. Briefly, 17 g of powder mixture (monopotassium phosphate, 22.66 g / L and sodium carbonate 7.78 g / L) was added to 400 mL of distilled water. After adding 0.5 mL Triton X-100, the mixture was dissolved on a magnetic stirrer at room temperature for 30 min. The final volume was made up to 500 mL in a graduated cylinder and used without further dilution. The buffer was stored at 4 °C until use. Ellman's reagent (5,5'-dithiobis-2-nitrobenzoic acid, DTNB, 0.5 mM) was freshly prepared for each AChE assay by weighing 2 mg into a 1.5 mL Eppendorf tube. It was dissolved in 1 mL of phosphate buffer (pH 7.2 with 0.1% triton X-100) by vortexing for 1-2 min. The final volume was brought to 10 mL in a 15 mL Falcon tube containing phosphate buffer (pH 7.2 with 0.1% triton X-100) and stored at 4 °C until use. Acetylcholine thioiodide (ATI, 5.76 mM) was freshly prepared for each AChE assay by weighing 2 mg into a 1.5 mL Eppendorf tube. It was dissolved in 1.2 mL of distilled water by vortexing for 1-2 min and stored at 4 °C until use.

[0168] AChE standard dilutions were prepared in phosphate buffer (pH 7.2 with 0.1% triton X-100) and used immediately. Ready-to-use 50 U / mL AChE stock (from Electrophorus electricus) was purchased from AAT Bioquest® Inc., Sunnyvale, CA, USA. It was diluted to prepare 1000 mU / mL AChE according to the manufacturer's instructions and further diluted in a 1:2 ratio to obtain eight different dilutions ranging from 4 to 500 mU / mL.

[0169] <Colorimetric measurement> To measure the activity of AChE, cells obtained by culturing in skeletal muscle differentiation medium according to Example 3 were treated as follows: Differentiation medium was carefully removed from the 24-well plate, and 300 μL of 0.5 mM DTNB solution (prepared in phosphate buffer, pH 7.2 with 0.1% triton X-100) was immediately added. After 2 minutes of incubation at room temperature in the dark, 50 μL of 5.76 mM ATI (prepared in distilled water) was added. The reaction contents were incubated at 30° C. for 60 minutes in the dark, and then the OD at 412 nm was measured with an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK).

[0170] For AChE enzyme standard analysis, dilutions of AChE enzyme standard (AAT® Bioquest, Sunnyvale, USA) ranging from 500 to 4 mU / ml were prepared as above, 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 mixtures was measured in a 24-well plate for 60 min.

[0171] <AChE mU rel / mg-protein calculation> AChE mU rel was calculated based on the OD412 values ​​obtained from the 60 min colorimetric measurement of cells by extrapolation of a linear standard curve obtained from the AChE standard measurement (OD at 412 nm after 7 or 8 min). rel Values ​​are AChE mU / mg protein in mg of total protein (calculated according to Example 19) of the corresponding cells cultured in skeletal muscle differentiation medium. rel It was calculated by dividing.

[0172] [Creatine kinase activity measurement] To measure the activity of AChE, cells obtained by culturing in skeletal muscle differentiation medium according to Example 3 were treated as follows: The medium was gently removed from the cells grown on 24-well plates, and the cells were washed with 1 ml of Trirode's salt solution (Sigma-Aldrich Co. LLC, MO, USA). Immediately after, 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 dH2O (LC-MS-Ultrachromasol, Fluka). After 5 min of incubation at 4°C in the dark, 400 μl of CK-NAC (Thermo Scientific, MA, USA), pre-dissolved by adding 10 ml of dH2O, was added. The reaction was analyzed by measuring the OD absorbance at 340 nm in an Anthos Zenith 340rt microplate reader (Biochrom Ltd., Cambridge, UK) set at 30°C. Unless otherwise stated, OD340nm values ​​obtained 21 min after addition of CK-NAC were used for subsequent analysis. rel CK activity in was calculated according to the manufacturer's instructions and normalized by dividing by mg total protein of the corresponding cells unless otherwise stated.

[0173] The MPCs of Example 3 analyzed for AChE and CK activity according to this example were found to be AChE+ and CK+, whereas the iSMCs of the present invention (Example 3) were found to be AChE- and CK- (Figure 14).

[0174] Example 18 - Comparison of cells according to the invention with cells known in the art Thurner et al. 2018 described the isolation of SMDCs characterized as either CD56+ or CD56-. To compare the cells described by Thurner et al. 2018 with the cells of the present invention, the authors of the referenced study were contacted and asked to provide samples. The authors agreed to provide cells isolated according to Thurner et al. 2018, and therefore these cells could be tested according to examples 3, 4, 5, 7, 8, 9, 16 and 17 of the present invention and compared to MPCs, MSCs, MPC-iSMCs and MSC-iSMCs isolated according to example 1. The results are shown in Figure 15.

[0175] Frudinger et al. 2018 described the isolation of SMDCs characterized as CD56+. To compare the cells described by Frudinger et al. 2018 with the cells of the present invention, the authors of the referenced study were contacted and asked to provide samples. The authors agreed to provide cells isolated according to Frudinger et al. 2018, so that these cells could be tested according to examples 3, 4, 5, 7, 8, 9, 16 and 17 of the present invention and compared to MPCs, MSCs, MPC-iSMCs and MSC-iSMCs isolated according to example 1. The results are shown in Figure 15.

[0176] Example 19 - Protein quantification Cells obtained by culturing in skeletal muscle differentiation medium according to Example 3 were analyzed for total protein quantification according to Thurner et al. 2018. Thus, the attached cells were first washed twice with PBS, followed by covering with PBST (0.1% Triton X-100) and then incubated at room temperature for 10 min. The lysates were then resuspended and transferred to Eppendorf® tubes, vortexed briefly, and then centrifuged at 1200×g for 4 min. Finally, the clear supernatant was transferred to a new Eppendorf tube and the protein concentration was measured by measuring the OD at 540 nm on an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK) using the Pierce BCA Protein Analysis Kit (Thermo Scientific, MA, USA) according to the manufacturer's instructions.

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Claims

1. 1. An in vitro or ex vivo method for obtaining induced smooth muscle cells (iSMCs), comprising: (a) isolating skeletal muscle-derived cells from skeletal muscle tissue obtained from a subject; (b) transdifferentiating the skeletal muscle-derived cells to obtain iSMCs by culturing the cells in a medium containing TGF-beta and heparin for 3 to 6 days. Including, Skeletal muscle-derived cells are (i) being mesenchymal oligopotent cells committed to the myogenic lineage; and (ii) positive expression of CD56 and desmin, and negative expression of CD34 and aSMA The myogenic progenitor cell (MPC) is characterized by:

2. The method of claim 1, wherein the TGF-beta in the medium of step (b) is TGFb1, TGFb2 and / or TGFb3.

3. The method of claim 1 or 2, wherein the iSMCs obtained in step (b) are non-fusion competent and / or characterized by positive expression of aSMA, CD49a, and CD146.

4. The method of any one of claims 1 to 3, wherein the iSMCs obtained from the MPCs in step (b) are characterized by positive expression of aSMA, CD49a, desmin, CD56, and CD146, and negative expression of CD34.

5. The method according to any one of claims 1 to 4, wherein step (a) is followed by step (a1), which comprises expanding the skeletal muscle-derived cells.

6. Step (a1) is 20 to 40 x 10 6 The method of claim 5, wherein the method is performed to receive a single cell.

7. The method according to any one of claims 1 to 6, wherein step (b) is carried out in a cell culture medium containing 1 to 10 μg / ml TGFb1 and 10 to 30 μg / ml heparin or 1 to 6 U / ml heparin.

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