Treatment of erectile dysfunction using amniotic fluid-derived mesenchymal stem cells

Abstract

The present invention provides a method of treating disorders or conditions associated with erectile dysfunction, comprising administering to a subject in need thereof a therapeutically effective amount of mesenchymal stem cells or secretomes thereof, said mesenchymal stem cells or secretomes being derived from human amniotic fluid.

Description

[Technical field]

[0001] The present invention provides methods for treating and preventing individuals with erectile dysfunction using mesenchymal stem cells (MSCs) or their secretome. [Background technology]

[0002] Erectile function is a hemodynamic process of blood flow and pressure maintenance within the cavernous space. After sexual arousal, release of nitric oxide into the erectile tissue, three processes occur to achieve an erection. These are relaxation of the trabecular smooth muscle, arterial dilation, and venous compression. In the final stage, arterial flow fills the sinusoidal space and compresses the subcapsular veins, reducing venous outflow. Blood enters the cavernous space of the penis, thereby enlarging and stretching the penis into a rigid organ. Blood flow in and out of the cavernous space is controlled by cavernous smooth muscle cells (CSMCs) embedded in the trabeculae of the cavernous space.

[0003] Erectile dysfunction (ED) is a condition that affects one in ten men in his lifetime, and ED may be associated with other problems that interfere with sexual intercourse, such as lack of desire, orgasm, and ejaculation problems.

[0004] Currently, ED is treated with common oral medications such as sildenafil, vardenafil or tadalafil, which may or may not improve ED.However, people who take drugs containing nitrates cannot take these drugs, and these drugs may cause side effects such as low blood pressure, and these drugs have many adverse reactions, which may include, but are not limited to, vision problems and seizures.

[0005] Alternative treatment options for ED may include the use of penis pumps or penile prostheses. However, because the devices are placed during surgery, there is a high risk of complications such as infection, bleeding, urethral perforation, and penile numbness. Acting alone, most drugs, pumps, and implants do not adequately reverse ED, and the level of treatment failure is unacceptably high.

[0006] Cavernous nerve-sparing prostatectomy (CNSP) is a method for treating prostate diseases such as prostate cancer, which avoids cutting the nerves near the prostate. However, there are problems with erectile function recovery and the risk of infection after CNSP. In addition, in some cases, even if a very careful nerve-sparing method is used based on the differences in nerve tissue between different patients and a certain surgical method is perfectly performed, it cannot accommodate the anatomical structure of a particular patient, resulting in differences in erectile function recovery, and there is also a very high risk of causing damage and degeneration of the cavernous nerve.

[0007] Accordingly, there is a need for alternative systems and methods for the treatment and prevention of individuals with erectile dysfunction to overcome the above-mentioned shortcomings of the prior art. Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above shortcomings, the present disclosure provides a method for treating a subject having erectile dysfunction, comprising administering to the subject a therapeutically effective amount of mesenchymal stem cells (MSCs).Also provides a method for treating a subject having erectile dysfunction, comprising administering to the subject a therapeutically effective amount of mesenchymal stem cell-derived secretome. [Means for solving the problem]

[0009] In at least one embodiment of the present disclosure, the mesenchymal stem cells may be derived from amniotic fluid, bone marrow, umbilical cord blood, placental tissue, adipose tissue, peripheral blood, and dental pulp, but the present disclosure is not limited thereto. In some embodiments, the mesenchymal stem cells are preferably derived from human amniotic fluid.

[0010] In at least one embodiment of the present disclosure, the erectile dysfunction is caused by cardiovascular disease, diabetes, anatomic defects, neurological disorders, hormonal deficiencies, side effects of medications, or any combination thereof.

[0011] In at least one embodiment of the present disclosure, the erectile dysfunction is neurogenic erectile dysfunction.

[0012] In at least one embodiment of the present disclosure, the neurogenic erectile dysfunction is caused by stroke, brain and / or spinal cord injury, diabetes, multiple sclerosis, Parkinson's disease, trauma from radical prostatectomy or radical pelvic surgery, or any combination thereof.

[0013] In at least one embodiment of the present disclosure, the erectile dysfunction comprises smooth muscle-related, arterial dilation, venous compression, or nerve atrophy.

[0014] In at least one embodiment of the present disclosure, the therapeutically effective amount of mesenchymal stem cells is at least 1×10 6 In some embodiments, a therapeutically effective amount of mesenchymal stem cells derived from amniotic fluid is administered to improve smooth muscle-related, intracellular pressure, arterial dilation, venous compression, or neuronal atrophy in a dose of about 1×10 6 ~2×10 8 The dose is administered to the subject within the range.

[0015] In at least one embodiment of the present disclosure, the mesenchymal stem cells may be expanded in culture for at least 2 weeks, 3 weeks, or 4 weeks.

[0016] In at least one embodiment of the present disclosure, the mesenchymal stem cells are positive for CD73, CD90, CD105, Nestin, Sox2, or any combination thereof, and the mesenchymal stem cells are negative for CD34, CD45, CD14, CD11b, CD79α, CD19, HLA-DR, or any combination thereof.

[0017] In at least one embodiment of the present disclosure, the mesenchymal stem cells may have, but are not limited to, a spindle-shaped morphology, a plate-shaped morphology, or a fibroblast-like morphology in adherent culture.

[0018] In at least one embodiment of the present disclosure, the mesenchymal stem cells have osteogenic potential, adipogenic potential, chondrogenic potential, or any combination thereof.

[0019] In at least one embodiment of the present disclosure, the mesenchymal stem cells are (a) obtaining an amniotic fluid sample from first, second or third trimester amniotic fluid by amniocentesis or Caesarean section; (b) centrifuging the amniotic fluid sample at 200×g for at least 5 minutes and removing the supernatant; (c) culturing the cells in alpha modified minimal essential medium or commercially available mesenchymal stem cell medium supplemented with 1-5% human platelet lysate or 10-20% fetal bovine serum; (d) removing non-adherent cells after 2-4 days of culture and growing the adherent cells as colonies for the next 7-14 days; Trypsinize adherent cells and plate them at 1000–9000 cells / cm. 2 and (e) passage and expanding the cells at a seeding density of

[0020] In some embodiments of the present disclosure, the alpha modified minimal essential medium comprises 1-20 ng / ml basic fibroblast growth factor, for example, about 1, 4, 10 or 20 ng / ml basic fibroblast growth factor. In some embodiments of the present disclosure, the alpha modified minimal essential medium does not comprise basic fibroblast growth factor.

[0021] In at least one embodiment of the present disclosure, the secretome of a mesenchymal stem cell is (a) culturing amniotic fluid stem cells in a basal medium for about 24 to 72 hours until the cells reach about 80% confluence; (b) collecting the culture medium supernatant after centrifugation at about 300×g for about 10 minutes to remove dead cells and debris; (c) filtering the supernatant through a 0.22 μm filter and storing the secretome at about −20° C. Effect of the Invention

[0022] Stem cell therapy has been proposed for the treatment of ED, because the stem cells can differentiate into endothelial cells, neuronal cells, and smooth muscle cells to restore possible structural damage of the penile tissue. The use of mesenchymal stem cells obtained from amniotic fluid can improve the restoration of the target tissue of the penis by local or systemic injection, by regenerating and promoting the proliferation and differentiation of progenitor cells. Thus, the mesenchymal stem cells of amniotic fluid are used to improve or treat the symptoms of erectile dysfunction. Compared with the prior art, when the mesenchymal stem cells of the amniotic fluid disclosed herein are applied to a subject, the mesenchymal stem cells of amniotic fluid can differentiate into endothelial cells, neuronal cells, and smooth muscle cells to restore possible structural damage of the penile tissue, so that at least the erectile function, the actin expression of the smooth muscle of the cavernous tissue, the structure of the cavernous nerve, and the vWF expression of the cavernous tissue can be significantly improved, which is helpful in the treatment of ED. [Brief description of the drawings]

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0024] The present disclosure will be more readily understood with reference to the accompanying drawings and the following description.

[0025] [Figure 1A-1]Representative examples of intracavernosal pressure (ICP) and mean arterial pressure (BP) recordings in sham, bilateral cavernous nerve crushing (BCNC) injury, and human amniotic fluid stem cell (hAFSC) groups in response to electrical stimulation of the distal end of the cavernous nerve. The x-axis represents time (seconds) and the green bar represents a single electrical stimulation of 60 seconds. The y-axis represents ICP and BP (upper and lower panels) of experimental animals. [Figure 1A-2] Measured values ​​of maximum ICP (cmH2O) in the sham, BCNC and hAFSC groups. [Figure 1A-3] Measured values ​​of area under the curve (AUC (Area Under Curve), cmH2O*sec) for the sham, BCNC and hAFSC groups. [Figure 1A-4] Measured values ​​of mean arterial pressure (MAP, cmH2O) in the sham, BCNC and hAFSC groups. [Figure 1A-5] Measured values ​​of maximum ICP / MAP in the sham, BCNC and hAFSC groups. [Figure 1B-1] 1 is a representative example of ICP and mean arterial pressure (BP) recordings of sham, BCNC and hAFSC secretome-treated BCNC groups in response to electrical stimulation of the distal end of the cavernous nerve. The x-axis represents time (seconds) and the green bar represents one electrical stimulation of 60 seconds. The y-axis represents ICP and BP (upper and lower panels) of experimental animals. [Figure 1B-2] Measured maximum ICP (cmH2O) of the sham group, BCNC group, and BCNC group treated with hAFSC secretome. [Figure 1B-3] Measured values ​​of AUC(cmH2O*sec) for the sham group, BCNC group and BCNC group treated with secretome of hAFSCs. [Figure 1B-4]MAP (cmH2O) measurements for the sham group, BCNC group, and BCNC group treated with hAFSC secretome. [Figure 1B-5] Measured values ​​of maximum ICP / MAP in the sham group, BCNC group, and BCNC group treated with secretome of hAFSCs. [Figure 1C-1] 1 is a representative example of ICP and mean arterial pressure (BP) recordings in response to electrical stimulation of the distal end of the cavernous nerve in sham, CNSP and hAFSC-treated CNSP groups. The x-axis represents time (seconds) and the green bar represents one electrical stimulation of 60 seconds. The y-axis represents ICP and BP (upper and lower panels) of experimental animals. [Figure 1C-2] Measured maximum ICP (cmH2O) in the sham group, the CNSP group, and the CNSP group treated with hAFSC. [Figure 1C-3] Measured AUC(cmH2O*sec) for the sham, CNSP and hAFSC-treated CNSP groups. [Figure 1C-4] MAP (cmH2O) measurements in the sham group, CNSP group, and CNSP group treated with hAFSCs. [Figure 1C-5] Measured values ​​of maximum ICP / MAP in the sham group, CNSP group, and CNSP group treated with hAFSC. [Figure 1D-1] Representative examples of ICP and BP recordings of sham, CNSP and hAFSC secretome treated CNSP groups in response to electrical stimulation of the distal end of the cavernous nerve. The x-axis represents time (seconds) and the green bar represents one electrical stimulation of 60 seconds. The y-axis represents ICP and BP of experimental animals (upper and lower panels). [Figure 1D-2] Measured maximum ICP (cmH2O) for the sham group, the CNSP group, and the CNSP group treated with hAFSC secretome. [Figure 1D-3] Measured AUC(cmH2O*sec) for the sham group, CNSP group, and CNSP group treated with hAFSC secretome. [Figure 1D-4]MAP (cmH2O) measurements in the sham group, CNSP group, and CNSP group treated with hAFSC secretome. [Figure 1D-5] Measured values ​​of maximum ICP / MAP in the sham group, CNSP group, and CNSP group treated with secretome of hAFSCs. [Figure 1E-1] These are the measured values ​​of maximum ICP (cmH2O) for the sham group, BCNC group, BCNC group treated with hAFSC, and BCNC group treated with secretome of hAFSC. [Figure 1E-2] MAP (cmH2O) measurements for the sham group, BCNC group, BCNC group treated with hAFSCs, and BCNC group treated with hAFSC secretome. [Figure 1E-3] Measured values ​​of delta ICP (cmH2O) in the sham group, BCNC group, BCNC group treated with hAFSCs, and BCNC group treated with hAFSC secretome. [Figure 1E-4] These are the measured values ​​of maximum ICP / MAP in the sham group, BCNC group, BCNC group treated with hAFSCs, and BCNC group treated with secretome of hAFSCs. [Figure 1E-5] The AUC (cmH2O*sec) measurements are for the sham group, the BCNC group, the BCNC group treated with hAFSCs, and the BCNC group treated with the secretome of hAFSCs. [Figure 1E-6] Measured values ​​of delta ICP / MAP in the sham group, BCNC group, BCNC group treated with hAFSCs, and BCNC group treated with secretome of hAFSCs. [Figure 1F-1] Measured maximum ICP (cmH2O) for the sham group, the CNSP group, the CNSP group treated with hAFSCs, and the CNSP group treated with hAFSC secretome. [Figure 1F-2] MAP (cmH2O) measurements for the sham group, CNSP group, CNSP group treated with hAFSCs, and CNSP group treated with hAFSCs secretome. [Figure 1F-3]Measured delta ICP (cmH2O) in the sham group, the CNSP group, the CNSP group treated with hAFSCs, and the CNSP group treated with hAFSCs secretome. [Figure 1F-4] Measured values ​​of maximum ICP / MAP for the sham group, CNSP group, CNSP group treated with hAFSCs, and CNSP group treated with secretome of hAFSCs. [Figure 1F-5] Measured AUC(cmH2O*sec) for the sham group, the CNSP group, the CNSP group treated with hAFSCs, and the CNSP group treated with hAFSCs secretome. [Figure 1F-6] Measured delta ICP / MAP in the sham group, the CNSP group, the CNSP group treated with hAFSCs, and the CNSP group treated with hAFSCs secretome. [Figure 2A] Immunofluorescence staining of nNOS and β-III tubulin in the major pelvic ganglia of the sham, BCNC and hAFSC groups. [Figure 2B] Immunofluorescence staining of nNOS and β-III tubulin in the major pelvic ganglia of the sham, CNSP and hAFSC groups. Scale bar = 400 μm. [Figure 3A-1] Immunofluorescence staining of nNOS and β-III tubulin in the dorsal nerve of the penis from the sham, BCNC and hAFSC groups. Scale bar = 50 μm. [Figure 3A-2] The expression levels (%) of nNOS in the dorsal nerve of the penis in the sham, BCNC and hAFSC groups are shown. [Figure 3A-3] The expression levels (%) of β-III tubulin in the dorsal nerve of the penis in the sham, BCNC, and hAFSC groups are shown. [Figure 3A-4] The nNOS / β-III tubulin expression ratio in the dorsal nerve of the penis in the sham group, BCNC group, and hAFSC group. [Figure 3B] The expression levels (%) of nNOS in the dorsal nerve of the penis in the sham group, CNSP group, and hAFSC group are shown. [Figure 3C] The expression levels (%) of nNOS in the dorsal nerve of the penis in the sham group, CNSP group, and hAFSC secretome group are shown. [Figure 4A] Ultrastructural analysis of cavernous nerves in sham, BCNC and hAFSC groups performed by transmission electron microscopy (TEM). [Figure 4B] Ultrastructural analysis of cavernous nerves in sham, BCNC and hAFSC secretome groups performed by TEM. [Figure 4C] Ultrastructural analysis of cavernous nerves in sham, CNSP and hAFSC groups performed by TEM. [Figure 5A-1] A series of immunofluorescence staining images of vWF in the corpus cavernosum of the sham, BCNC and hAFSC groups. The characteristics of sham, BCNC and hAFSC are provided by staining of cell-specific marker vWF and combined immunofluorescence. vWF is represented in green and nuclei are labeled in blue. The strong staining is due to the presence of vWF in the corpus cavernosum. Scale bar = 100 μm. [Figure 5A-2] Graph showing quantification of vWF expression (%) in the corpus cavernosum of the sham, BCNC and hAFSC groups. [Figure 5B-1] A series of immunofluorescence staining images of vWF in the corpus cavernosum of the sham, CNSP and hAFSC groups. Scale bar = 100 μm. [Figure 5B-2] 1 is a graph showing quantification of vWF expression in the corpus cavernosum of the sham, CNSP and hAFSC groups. [Figure 5C-1] A series of immunofluorescence staining images of vWF in the corpus cavernosum of the sham, CNSP and hAFSC secretome groups. The characteristics of the sham, CNSP and hAFSC secretomes are provided by staining for the cell-specific marker vWF (light green) and combined immunofluorescence. The hAFSC secretome group had significantly higher expression of vWF compared to the CNSP group. Scale bar = 100 μm. [Figure 5C-2] Graph showing quantification of vWF expression (%) in corpus cavernosum in sham, CNSP and hAFSC secretome groups. [Figure 6A-1] A series of immunofluorescence staining images of α-smooth muscle actin expression in the corpus cavernosum of the sham, BCNC and hAFSC groups. Scale bar = 400 μm. [Figure 6A-2] Measurement of α-smooth muscle actin (α-SMA) expression in the corpus cavernosum of the sham, BCNC and hAFSC groups. [Figure 6B-1] A series of immunofluorescence staining images of α-smooth muscle actin expression in the corpus cavernosum of the sham, CNSP and hAFSC groups. Scale bar = 400 μm. [Figure 6B-2] Measurement of α-SMA expression in the corpus cavernosum of the sham, CNSP and hAFSC groups. [Figure 6C-1] A series of immunofluorescence staining images of α-smooth muscle actin in the corpus cavernosum of the sham group, the CNSP group, and the hAFSC secretome group. Scale bar = 400 μm. [Figure 6C-2] Measurement of α-SMA expression in the corpus cavernosum of the sham, CNSP and hAFSC secretome groups. [Figure 7A] Ultrastructural analysis of corpus cavernosum (CC) tissues of sham, BCNC and hAFSC groups performed by transmission electron microscopy (TEM). As evident from the TEM images, the muscular layer is thicker and denser in the sham and hAFSC groups compared to the BCNC group. Scale bar = 5 μm. [Figure 7B] Ultrastructural analysis of corpus cavernosum (CC) tissues in the sham, CNSP and hAFSC groups performed by TEM. As evident from the TEM images, the muscular layer in the sham and hAFSC groups is thicker and more dense than that in the CNSP group. Scale bar = 5 μm. [Figure 7C] Ultrastructural analysis of corpus cavernosum (CC) tissues in the sham, CNSP and hAFSC secretome groups performed with TEM. As evident from the TEM images, the muscular layer is thicker and more dense in the sham and hAFSC secretome groups compared to the CNSP group. Scale bar = 5 μm. [Figure 8A-1] The measurements of maximum ICP (cmH2O) of different concentrations of hAFSCs including 2*104 hAFSCs, 2*105 hAFSCs, 2*106 hAFSCs, 1*107 hAFSCs and 2*107 hAFSCs in the sham group, BCNC group and hAFSC group. [Figure 8A-2]Measurement of area under the curve (AUC, cmH2O*sec) for different concentrations including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC in sham, BCNC and hAFSC groups. [Figure 8A-3] The changes of ICP (cmH2O) in different concentrations of sham group, BCNC group and hAFSC group, including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC. [Figure 8A-4] Measured mean arterial pressure (MAP, cmH2O) of different concentrations of hAFSCs including 2*104 hAFSCs, 2*105 hAFSCs, 2*106 hAFSCs, 1*107 hAFSCs and 2*107 hAFSCs in sham, BCNC and hAFSC groups. [Figure 8A-5] The measurements of maximum ICP / MAP of sham group, BCNC group and hAFSC group at different concentrations including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC. [Figure 8A-6] The changes of ICP / MAP in sham group, BCNC group and hAFSC group at different concentrations including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC. [Figure 8A-7] 1 is a representative example of the recording of intracavernous pressure (ICP) and mean arterial pressure (BP) of sham group, bilateral cavernous nerve crush (BCNC) injury group and human amniotic fluid stem cell (hAFSC) group in response to electrical stimulation of the distal end of cavernous nerve with different concentrations including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC. The x-axis represents time (seconds) and the green bar represents one electrical stimulation of 60 seconds. The y-axis represents the ICP and BP of experimental animals (upper and lower panels). [Figure 9]H&E staining and Masson's trichrome staining of cavernous nerve samples with different concentrations, including 2*104 hAFSCs, 2*105 hAFSCs, 2*106 hAFSCs, 1*107 hAFSCs and 2*107 hAFSCs, from sham, BCNC and hAFSC groups. Scale bar=400 μm. [Figure 10] Figure 1 shows the smooth muscle collagen ratios of different concentrations of sham, BCNC and hAFSC groups, including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC. Compared with sham group, p<0.05*, and compared with BCNC group, p<0.05#. [Figure 11] Immunofluorescence staining images of nNOS, β-III tubulin, and NF-1 provided to the dorsal nerve of the penis at different concentrations including 2*104 hAFSCs, 2*105 hAFSCs, 2*106 hAFSCs, 1*107 hAFSCs, and 2*107 hAFSCs in the sham, BCNC, and hAFSC groups. Scale bar=50 μm. nNOS is represented in green. β-III tubulin and NF-1 are represented in red, and nuclei are labeled in blue. [Figure 12] The nNOS / β-III tubulin expression ratios of the dorsal nerve of the penis at different concentrations, including 2*104 hAFSCs, 2*105 hAFSCs, 2*106 hAFSCs, 1*107 hAFSCs and 2*107 hAFSCs in the sham group, BCNC group and hAFSC group. [Figure 13] A series of immunofluorescence staining images of α-SMA and vWf of different concentrations of corpus cavernosum including 2*104 hAFSCs, 2*105 hAFSCs, 2*106 hAFSCs, 1*107 hAFSCs and 2*107 hAFSCs from sham group, BCNC group and hAFSC group. α-SMA (smooth muscle actin) is shown in red. vWf is shown in green and nuclei are labeled in blue. Scale bar of α-SMA=200 μm. Scale bar of vWF=100 μm. [Figure 14]Measurement of α-SMA expression in the corpus cavernosum in different concentrations of sham, BCNC and hAFSC groups, including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC. [Figure 15] The vWf expression in different concentrations of corpus cavernosum including 2*104 hAFSC, 2*105 hAFSC, 2*106 hAFSC, 1*107 hAFSC and 2*107 hAFSC in sham group, BCNC group and hAFSC group is measured. Compared with sham group, p<0.05*, and compared with BCNC group, p<0.0#. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The following embodiments are provided to describe the present disclosure in detail. Those skilled in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure of this specification, and can also implement or apply it in other different embodiments. Therefore, the following modes for implementing the present disclosure can be changed and / or modified without departing from the scope of different aspects and applications, and any element or method within the scope of the present disclosure disclosed herein can be combined with any other element or method disclosed in any embodiment of the present disclosure.

[0027] As used herein, the singular forms "a," "an," and "the" include plural referents unless specifically and clearly limited to one referent. The term "or" is used synonymously with the term "and / or" unless the context clearly dictates otherwise.

[0028] The numerical ranges used herein are inclusive and combinable, and any numerical value falling within the numerical ranges herein can be taken as a maximum or minimum value, from which subranges can be derived. For example, the numerical range "10-20%" is understood to include any subrange between the minimum value of 10% and the maximum value of 20%, such as the subranges of 10%-15%, 15%-20%, and 12.5%-17.5%.

[0029] As used herein, the term "about" when referring to a numerical value is meant to include a variation of ±20%, ±10%, ±5%, ±1%, ±0.5% or ±0.1% from the numerical value. Such variation in numerical value can occur, for example, due to experimental error, typical errors in the measuring or handling procedures for manufacturing the compounds, compositions, concentrates or formulations, differences in the source, preparation or purity of the starting materials or components used in the present disclosure, or similar considerations.

[0030] As used herein, a "subject" includes, but is not limited to, vertebrates such as humans, mammals, reptiles, amphibians, and / or fish. Advantageously, however, the subject is a mammal, such as a human, or a domesticated mammal, such as a dog, cat, horse, rat, mouse, or a livestock mammal, such as a cow, sheep, or pig.

[0031] As used herein, the terms "comprise," "comprising," "include," "including," "have," "having," "contain," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, if an object is described as "comprising" one limitation, unless otherwise specified, it may additionally include other ingredients, elements, components, structures, regions, parts, devices, systems, steps, or connections, etc., and should not exclude other limitations.

[0032] As used herein, "erectile dysfunction" refers to the inability to achieve or maintain an erection sufficient for satisfactory sexual performance.The etiological factors of neurogenic ED may include, but are not limited to, stroke, brain and spinal cord injury, type I and type II diabetes, chronic renal failure, chronic liver failure, central nervous tumor, multiple sclerosis, Parkinson's disease, or radical pelvic surgery.

[0033] As used herein, "administering" or "administration" or "injection" or "providing" refers to a technique for delivering a substance, such as stem cells or mesenchymal stem cells, to the body systemically or locally or any combination thereof. When a therapeutically effective amount of the present invention is administered parenterally or intravenously, it is usually prepared in a unit dose injection form (e.g., solution, suspension or emulsion). Pharmaceutical formulations suitable for injection may include sterile aqueous solutions or dispersions, sterile powders for reconstitution into sterile injectable aqueous solutions or dispersions.

[0034] As used herein, a "therapeutically effective amount" can be administered to a male, transgendered male, or male-like subject (e.g., to the cavernous nerve of the penis of a male subject) to treat erectile dysfunction (e.g., promote erection) within less than 60 minutes, less than 45 minutes, less than 30 minutes, or less than 15 minutes, or less than 5 minutes of application.

[0035] As used herein, the term "therapeutically effective amount" also refers to an amount of amniotic fluid stem cells or human amniotic fluid stem cells that, when administered to an individual to treat a condition, disease, disorder, or symptom caused by or associated with cavernous nerve injury, is sufficient to effect such treatment. The therapeutically effective amount varies depending on the particular condition, disease, disorder, or symptom being treated and its severity, as well as the age, weight, health, and responsiveness of the subject being treated. Thus, one or more of these parameters can be selected and adjusted to provide a therapeutically effective amount of amniotic fluid stem cells or human amniotic fluid stem cells. A therapeutically effective amount or therapeutically effective amount of hAFSCs is 1*10 5 For example, 1*10 6 , 1*10 7 , 1*10 8 , 1*10 9 , 2*10 1 , 2*10 2 , 2*10 3 , 2*10 4 , 2*10 5 , 1*10 6 , 2*10 7 , 2*108 Or more.

[0036] As used herein, the terms "paracrine secretome" or "secretome" or "stem cell-derived secretome" or "stem cell-derived paracrine secretome" or "mesenchymal stem cell-derived secretome" or "hAFSC secretome" are used interchangeably. In certain aspects, the secretome contains lipids, proteins, RNA, and microRNA, and the MSC-secreted secretome is involved in cardioprotective paracrine effects. In some embodiments of the present application, the MSC-derived secretome can enhance angiogenesis, viability, or proliferation. Furthermore, the secretome can prevent vascular injury and repair, for example, cardiac trauma, prostate injury, liver injury, or other organ injury.

[0037] As used herein, "vWF," "vWF factor," or "von Willebrand factor" is a clinical indicator of risk associated with atherosclerosis or an endothelial cell indicator.

[0038] Mesenchymal stem cells (MSCs) are transplanted into the cavernous nerves attached to the corpus cavernosum, migrate to the target area, and differentiate into endothelial and smooth muscle cells, followed by proliferation to form structures including myocardium, coronary arteries, arterioles, and capillaries, restoring erectile function in various subjects.

[0039] In at least one embodiment, the invention extends to, but is not limited to, stem cells or amniotic fluid stem cells derived from non-embryonic animal cells or tissues, capable of self-renewal and capable of differentiation into endodermal, ectodermal and mesodermal cells.

[0040] In a particular aspect, the present invention extends to, but is not limited to, mesenchymal stem cells derived from prenatal animal cells or tissues or amniotic fluid, capable of self-renewal and differentiation into endodermal, ectodermal and mesodermal cells.

[0041] As used herein, "corpus cavernosum" refers to one of the major tissue components of penile erectile tissue, which may include, but is not limited to, smooth muscle and endothelial cells.

[0042] As used herein, "cavernous nerve" refers to the nerve that facilitates penile erection, and originates from the pelvic splanchnic plexus and lies in the prostatic plexus. The cavernous nerve contains both sympathetic and parasympathetic fibers that originate from the pelvic plexus, and leaves the pelvis between the transversus pubis muscle and the membranous part of the urethra before passing under the pubic arch to supply each of the cavernous nerves, which supply the corpus cavernosum and the penile urethra, and terminate in a delicate meshwork around the erectile tissue, including, but not limited to, the corpus cavernosum.

[0043] As used herein, "amniocentesis" may be a procedure utilized to obtain 1-40 mL, e.g., 2-5 mL, 5-10 mL, 10-20 mL, 20-30 mL, or 30-40 mL, of amniotic fluid from a female subject by inserting a long subarachnoid puncture needle with a sharp cutting tip through the surface of the skin into the uterine cavity and obtaining the amniotic fluid by suction.

[0044] As used herein, "medium", "basal medium" or "media" refers to an optimal culture medium for the culture of a variety of animal cells, including, but not limited to, neural cells, stem cells, mesenchymal stem cells, primary epithelial cells, keratinocytes, cervical epithelial cells, renal epithelial cells and established cell lines. In at least one embodiment, the culture may be expansion or differentiation.

[0045] Furthermore, it is noted that, as used in this disclosure, "cavernous nerve injury" is used interchangeably with the term "cavernous nerve crush" and may include, but is not limited to, transection, resection, freezing, and crushing, unless the context clearly indicates otherwise. In one aspect of penile smooth muscle contraction, and in particular the molecular mechanisms of penile smooth muscle contraction, penile smooth muscle contraction and relaxation are mediated by cytoplasmic free Ca 2+Norepinephrine from nerve endings and endothelin, and prostaglandin F2α from the endothelium activate receptors in smooth muscle cells to increase inositol trisphosphate and diacylglycerol, releasing calcium from intracellular stores such as the sarcoplasmic reticulum and / or opening calcium channels in the smooth muscle cell membrane, allowing calcium to flow in from the extracellular space. This results in the release of cytoplasmic free Ca 2+ triggers a transient increase in Ca from resting levels, e.g., to 120 nM–270 nM–500 nM–700 nM. Furthermore, at elevated levels, 2+ Calmodulin binds to the myosin light chain kinase, changing its conformation to expose an interaction site with the myosin light chain kinase. The resulting activation catalyzes the phosphorylation of myosin light chains, triggering the cycling of myosin crossbridges (heads) along the actin filament and the generation of force. The phosphorylation of the light chain also activates myosin ATPase, which hydrolyzes ATP to energize muscle contraction (Dean RC et al., Urol Clin North Am, 32(4):379, 2005).

[0046] As used herein, "nerve-sparing radical prostatectomy" involves severing the nerve bundles from the side of the prostate.

[0047] As used herein, "sham" or "sham surgery" refers to a control group or placebo.

[0048] The MSCs of the present invention may be isolated from non-embryonic tissue selected from the group consisting of muscle, dermis, fat, tendon, ligament, perichondrium, periosteum, heart, aorta, endocardium, myocardium, epicardium, aorta and vein, granulation tissue, peripheral nerves, peripheral ganglia, spinal cord, dura mater, pia mater, trachea, esophagus, stomach, small intestine, large intestine, liver, spleen, pancreas, parietal peritoneum, visceral peritoneum, parietal pleura, visceral pleura, bladder, corpus cavernosum, spongiosum, urethra, gallbladder, kidney, placental tissue, acellular amniotic membrane, amniotic fluid, connective tissue or bone marrow.

[0049] In at least one embodiment of the present disclosure, ED is caused by multiple chronic diseases and factors, including, but not limited to, vascular disease, stroke, diabetes, hormone deficiency (e.g., hypogonadism), neurological disorders (e.g., Parkinson's disease and trauma from radical prostatectomy), multiple sclerosis, radical pelvic surgery, psychiatric conditions, trauma including brain and spinal cord injury. In some embodiments of the present disclosure, neurogenic ED is a common complication after radical prostatectomy for prostate cancer. In some embodiments of the present disclosure, the subject suffers from ED as a side effect of administration of certain drugs, including, but not limited to, diuretics, antihypertensives, antihistamines, antidepressants, Parkinson's drugs, antiarrhythmics, tranquilizers, muscle relaxants, nonsteroidal anti-inflammatory drugs, histamine H2 receptor antagonists, hormones, chemotherapy drugs, prostate cancer drugs, anti-seizure drugs, etc.

[0050] In at least one embodiment of the present disclosure, high intracavernous blood pressure (ICP) is maintained by a low inflow rate.

[0051] In at least one embodiment of the present disclosure, ED may be improved by increasing blood flow to the penis by enhancing the effects of nitric oxide, which causes increased blood flow.

[0052] In at least one embodiment of the present disclosure, the underlying mechanism of ED may be vascular, neurogenic, anatomical, hormonal, pharmacological and / or psychogenic.

[0053] In at least one embodiment of the present disclosure, the MSCs of the present invention may be isolated from, but are not limited to, non-human cells, human cells, amniotic cells, and human ammonitic fluid.

[0054] These MSCs, complexes or secretomes may be used for a variety of applications including, but not limited to, the treatment or prevention of various organ or organ system failures such as heart failure, liver failure, or erectile dysfunction.

[0055] In at least one embodiment of the present disclosure, a method of treating erectile dysfunction may include providing, administering or giving animal amniotic fluid stem cells, human amniotic fluid stem cells, mesenchymal stem cells, umbilical cord blood stem cells, placental stem cells, bone marrow stem cells, adipose tissue-derived stem cells, and any other type of stem cells.

[0056] In at least one embodiment of the present disclosure, changes in penile tissue following CN injury may include decreased intracellular pressure, apoptosis of smooth muscle and endothelial cells, decreased neural-nitric oxide synthase (nNOS) nerve fiber density, upregulation of fibroproliferative cytokines (e.g., TGFβ1), or abnormal biological signal responses (e.g., ROS). In some embodiments, MSCs may be used, with approximately 1*10 6 , 1*10 7 , 1*10 8 , 1*10 9 , 2*10 1 , 2*10 2 , 2*10 3 , 2*10 4 , 2*10 5 , 2*10 6 , 1*10 6 , 2*10 7 or 2*10 8 The development of erectile dysfunction can be treated or prevented by providing an amount of amniotic fluid mesenchymal stem cells administered at 2×10 per side. In some embodiments, the dosage form of hAFSCs in saline is 2×10 per side. 5 There are 4 x 10 cells 5 Cells, 1 x 10 on each side 6 There are 2 x 10 cells 6 cells, or 5 x 10 on each side 6 There are 1 x 10 cells 7 Some embodiments of the present disclosure provide a method for administering to a subject a therapeutically effective amount of a plurality of mesenchymal stem cells, the plurality of MSCs being obtained from at least amniotic fluid. The MSCs can improve, but are not limited to, smooth muscle association, arterial dilation, venous compression, and neuronal atrophy.

[0057] Corpus cavernosum tissue was harvested and subjected to histological analysis. Various analytical methods may include, for example, hematoxylin-eosin staining, H&E staining, Masson's trichrome staining, collagen staining and Roxarco Luxol fast blue staining, Western blotting, immunofluorescence analysis to examine the expression of specific proteins (α-SMA, eNOS, nNOS, iNOS, beta-III tubulin, NF-1 and von Willebrand factor), terminal deoxyribonucleotidyl transferase-mediated nick end labeling analysis (TUNEL assay), reverse transcription and quantitative real-time polymerase chain reaction (PCR) to evaluate fibrosis gene expression. EXAMPLES

[0058] The following examples further describe exemplary embodiments of the present disclosure and should not be construed as limiting the scope of the disclosure.

[0059] Example 1: Animal model and experimental design Twelve-week-old male Sprague-Dawley rats were obtained from BioLasco, Taiwan. All rats were housed under standard laboratory conditions, and animal studies were reviewed and approved by the Fu Jen University Animal Care and Use Committee.

[0060] For surgery, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg). After the abdomen was shaved and prepared with iodine solution, a lower midline abdominal incision was made. The prostate was then exposed, and the bilateral posterolateral CN and major pelvic ganglion (MPG) were identified. Sham-operated rats had no further surgical procedures. In the BCNC group, the CN was isolated and a crush injury was applied for 2 min using hemostats (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD), and the abdomen was closed with two layers of sutures. This procedure resulted in the BCNC injury. In the nerve-sparing radical prostatectomy (CNSP) group, minimal transection of the nerves was involved in intrafascial and extrafascial dissection.

[0061] Human amniotic fluid mesenchymal stem cells (hAFMSCs) were produced, for example, by a two-step culture protocol including culture of human amniotic cells and culture of mesenchymal stem cells based on the method of harvesting mesenchymal stem cells from human amniotic fluid. For culturing human amniotic cells, for example, primary amniocyte culture was used according to a predetermined or standard amniotic cell culture protocol performed in a cytogenetic laboratory (US7101710B2).

[0062] The method for collecting mesenchymal stem cells from human amniotic fluid includes culturing (a) a plurality of human amniotic fluid amniocytes and culturing (b) a plurality of mesenchymal stem cells, wherein the culturing (a) of the human amniotic fluid amniocytes includes a step (i) of constructing a primary amniotic cell culture comprising a plurality of adherent human amniotic fluid cells and a supernatant containing a plurality of non-adherent human amniotic fluid cells and a liquid medium, and a step (ii) of recovering the non-adherent human amniotic fluid cells in the supernatant, and the culturing (b) of the mesenchymal stem cells includes a step (i) of centrifuging the non-adherent human amniotic fluid cells, a step (ii) of seeding the non-adherent human amniotic fluid cells centrifuged in step (i) of b in an alpha modified minimal essential medium supplemented with fetal bovine serum in a culture bottle, and a step (iii) of incubating the non-adherent human amniotic fluid cells seeded in step (ii) of b in humidified CO2 to promote the growth of the mesenchymal stem cells.

[0063] In a method for collecting secretomes from human amniotic fluid, the secretomes are prepared by a method including the steps of culturing amniotic fluid stem cells in a basal medium for 24-72 hours until 80% confluence, and collecting the culture medium supernatant after centrifugation at 300xg for 10 minutes to remove non-permanently damaged cells, permanently damaged cells, dead cells and debris. The supernatant was then filtered through a 0.22μm filter and stored at -20℃.

[0064] Example 2: Treatment of ED caused by BCNC by administration of human amniotic fluid stem cells The experimental design and surgical procedure were performed as described in Example 1. Specifically, rats were randomly assigned to three groups: a sham group (n=8), a bilateral cavernous nerve crush (BCNC) injury group (n=8), and a BCNC group treated with hAFSCs (n=8). After collecting mesenchymal stem cells from human amniotic fluid, approximately 1×10 cells were collected from 200 μL of hAFSCs. 6The cells were administered from the injury site. As shown in FIG. 1A-1, injection according to the present invention, for example, intracavernous injection of hAFSC, resulted in ICP recovery-time curves, suggesting spontaneous nerve regeneration. The maximum ICP of the hAFSC group was significantly higher than that of the BCNC group. As shown in FIG. 1A-2 to 1A-5, the hAFSC group in all figures was significantly higher than the BCNC group, except for FIG. 1A-4, where the mean arterial pressure (MAP) remained in the normal range. Accordingly, an embodiment of the present invention provides a method for delivering an effective therapy to a subject to treat or prevent erectile dysfunction.

[0065] Example 3: Treatment of ED caused by BCNC by administration of hAFSC secretome The experimental design and surgery are performed as described in Example 1, but with administration of hAFSC secretome. Specifically, rats were randomly assigned to three groups: sham group (n=8), BCNC group (n=8), and BCNC injury group treated with hAFSC secretome (n=8). After BCNC injury, 200 μL of hAFSC secretome was administered four times a week from the injury site. As shown in FIG. 1B-5, the maximum ICP / MAP of the BCNC injury group treated with hAFSC secretome according to the present invention shows a higher ratio than that of the BCNC group. Accordingly, an embodiment of the present invention provides a method for delivering hAFSC secretome to the injury site to deliver effective treatment to a target for treating or preventing erectile dysfunction.

[0066] Example 4: Treatment of ED caused by CNSP by administration of human amniotic fluid stem cells The experimental design and surgical procedure were performed as described in Example 1. The rats were randomly assigned to three groups: sham group (n=8), CNSP group (n=8), and CNSP group treated with hAFSC (n=8). After collecting mesenchymal stem cells from human amniotic fluid, approximately 1×10 6 cells were cultured in 200 μL of hAFSC. 6The cells were administered from the site of injury caused by CNSP. As shown in FIG. 1C-1, injection according to the present invention, for example, intracavernosal injection of hAFSC, resulted in an ICP recovery-time curve, in which the maximum ICP of the hAFSC group was significantly higher than that of the CNSP group. As shown in FIG. 1C-5, the maximum ICP / MAP of the hAFSC group according to the present invention is significantly higher than that of the CNSP group. Accordingly, an embodiment of the present invention provides a method for delivering an effective therapy to a subject to treat or prevent erectile dysfunction.

[0067] Example 5: Treatment of ED due to CNSP by administration of hAFSC secretome The rats were randomly assigned to three groups: sham group (n=8), CNSP group (n=8), and CNSP group treated with hAFSC secretome (n=8). After CNSP injury, 200 μL of hAFSC secretome was administered four times a week from the injury site. As shown in FIG. 1D-1, the injection according to the present invention, for example, intracavernous injection of hAFSC secretome, resulted in an ICP recovery-time curve, and the maximum ICP of the secretome group was significantly higher than that of the CNSP group. As shown in FIG. 1D-2 to 1D-5, the maximum ICP / MAP of the CNSP group treated with hAFSC secretome shows a significantly higher ratio than that of the CNSP group.

[0068] Also, as shown in FIG. 1E-1, the rat group with ED caused by BCNC treated with hAFSC or secretome showed significantly higher maximum ICP, i.e., about 100 cmH2O pressure, compared with the BCNC group. As shown in FIG. 1E-6, the delta ICP / MAP ratio of the ED caused by BCNC treated with hAFSC or secretome group was greater than 0.5, which was significantly higher than that of the BCNC group. Accordingly, an embodiment of the present invention provides a method for delivering an effective therapy to a subject to treat or prevent erectile dysfunction.

[0069] Similarly, as shown in Figure 1F-1, the CNSP-induced ED group treated with hAFSCs or secretome showed a significantly higher maximum ICP, i.e., about 100 cmH2O pressure, than the CNSP group. As shown in Figure 1F-6, the delta ICP / MAP ratio of the CNSP-induced ED group treated with hAFSCs or secretome was greater than 0.5, significantly higher than that of the CNSP group, which was less than 0.2.

[0070] Example 6: Immunofluorescence staining of the dorsal nerve of the penis The ultrastructural changes of cavernous tissue, such as changes in mitochondria, were observed using a transmission electron microscope.The efficacy and preliminary effective mechanism of amniotic fluid stem cells in treating cavernous nerve injury were confirmed after statistical analysis.

[0071] As shown in Figure 2A and Figure 2B, immunofluorescence staining of nNOS and β-III tubulin was demonstrated and expressed in the nerve fibers of the dorsal nerve of the penis in the sham, BCNC and hAFSC groups. The expression of nNOS in the dorsal nerve of the penis in the BCNC group was significantly lower than that in both the sham and hAFSC groups (p<0.05). As shown in immunofluorescence staining in Figure 3A-1, β-III tubulin immunostaining was performed on the nNOS-positive nerve fibers of the dorsal nerve of the penis to identify the nerve fibers positive for nNOS and to quantify their nNOS. As shown in Figure 3A-4, the area ratio of nNOS / β-III tubulin expression in the BCNC group was significantly lower than that in the sham and hAFSC groups (p<0.05). Moreover, ultrastructural analysis of the cavernous nerve in Figure 7A demonstrated that the circular nerve fibers and myelin debris in the BCNC group were visually smaller than those in the sham and hAFSC groups. Similarly, ultrastructural analysis of the cavernous nerve in Figure 7B demonstrated that the circular nerve fibers and myelin debris in the CNSP group were visually smaller than those in the sham and hAFSC groups. Similarly, ultrastructural analysis of the cavernous nerve in Figure 7C demonstrated that the circular nerve fibers and myelin debris in the CNSP group were visually smaller than those in the sham and secretome groups. Treatment with secretome or hAFSC reduced the amount of myelin debris, myelin began to regenerate, and a thick myelin sheath was observed.

[0072] Example 7: Immunofluorescence staining of corpus cavernosum Immunofluorescence expression of vWF shown in Figure 5A-1 and α-SMA shown in Figure 6A was also shown in the sham, BCNC and hAFSC groups. The vWF expression in the BCNC and hAFSC groups was significantly lower than that in the sham group (p<0.05), while the BCNC group was significantly different from the hAFSC group (p<0.05), and the BCNC group was significantly different from the hAFSC group (p<0.05). Meanwhile, the immunofluorescence expression of α-SMA in the BCNC group only shows a significant difference compared to both the sham and hAFSC groups. The integrity of the cavernous tissue was also shown in the ultrastructural analysis of the cavernous nerve (Figure 7A), which demonstrated that the sham and hAFSC groups had a thicker and denser muscle layer compared to the BCNC group.

[0073] statistical analysis Data are expressed as mean ± standard deviation. Differences between the means of treatment groups were detected by analysis of variance (ANOVA) and Scheffe post hoc test, and statistical significance was determined at P < 0.05. Statistical analysis was performed using SPSS 12.0 for Windows (SPSS, Inc., Chicago).

[0074] The above detailed description of the embodiments is intended to illustrate the preferred implementations of the present disclosure, and is not intended to limit the scope of the present disclosure. Accordingly, all modifications and changes made by those skilled in the art should be included within the scope of the present disclosure as defined by the appended claims.

[0075] Example 8: Therapeutic Effective Dose of Human Amniotic Fluid Stem Cells for Bilateral Cavernous Nerve Crush Injury The experimental design and surgical procedures were performed as described in Example 1, but with different concentrations of hAFSCs, e.g., 2*10 4 hAFSC, 2*10 5 hAFSC, 2*10 6 hAFSC, 1*10 7 hAFSC and 2*10 7Specifically, rats were divided into a sham group (n=8), a bilateral cavernous nerve crush injury (BCNC) group (n=7), and a 2*10 hAFSC group. 4 BCNC group (n=5) treated with hAFSCs at a dose of 2*10 5 (n=5), 2*10 6 (n=14), 1*10 7 (n=6), and 2*10 7 The subjects were randomly assigned to one of seven groups: the BCNC group treated with hAFSCs (n=7). After harvesting mesenchymal stem cells from human amniotic fluid, approximately 2*10 4 , 2*10 5 , 2*10 6 , 1*10 7 and 2*10 7 The cells were administered to each group through the injury site. As shown in Figure 8A-7, the injection according to the present invention, for example, the intracavernous injection of hAFSCs, resulted in an ICP recovery-time curve, which suggested spontaneous nerve regeneration, and the recovery effect was 2*10 5 In other words, the therapeutically effective dose of hAFSCs to treat nerve injury, e.g., bilateral cavernous nerve crush, is highest in the hAFSC group with a cell dose of 2*10 5 The maximum ICP of the hAFSC group at all concentrations tested was significantly higher than that of the BCNC group. As shown in Figures 8A-1 to 8A-6, the maximum ICP of the hAFSC group was significantly higher than that of the BCNC group at all concentrations tested. 4 In addition to the lower mean arterial pressure (MAP) of the hAFSC group than that of the BCNC group (Figure 8A-4), the hAFSC group was significantly higher than the BCNC group. Figure 10 shows that the smooth muscle to collagen ratio was 2*10 510 hAFSC group, which is consistent with and reflected by H&E staining and Masson staining in FIG. 9. Accordingly, an embodiment of the present invention provides a therapeutically effective amount to deliver effective therapy to a subject to treat or prevent erectile dysfunction. In FIG. 11, β-III tubulin immunostaining was performed on nerve fibers of the dorsal nerve of the penis to identify fibers positive for nNOS and quantify their nNOS content. Quantitative analysis in FIG. 12 showed that the number of nerve fibers positive for nNOS in the BCNC group was dramatically reduced compared to the sham group, while the number of nerve fibers positive for nNOS in all hAFSC groups at different concentrations was significantly increased compared to the BCNC group. In addition, the number of nerve fibers positive for nNOS was 2*10 5 and 2*10 6 The increase was most significant in the hAFSC group, which was consistent with FIG. 11 and reflected in the immunofluorescence staining of nNOS, β-III tubulin, and NF-1 in the dorsal nerve of the penis in FIG.

[0076] Furthermore, the smooth muscle cell content in the corpus cavernosum was evaluated by α-smooth muscle actin staining, and the smooth muscle cell content in the corpus cavernosum of the BCNC group was significantly lower than that of the sham group and the group treated with hAFSCs. As shown in Figure 14, the number of α-smooth muscle actin was 2*10 5 and 2*10 6 hAFSC group, which was consistent with and reflected in the immunofluorescence staining images in FIG. 13. Similarly, as shown in FIG. 15, the number of vwf expression was 2*10 5 and 2*10 6 hAFSC group, which was consistent with FIG. 13 and reflected in the immunofluorescence staining images in FIG.

Claims

1. 1. A method of treating erectile dysfunction, comprising administering to a subject in need thereof a therapeutically effective amount of mesenchymal stem cells, wherein said mesenchymal stem cells are derived from amniotic fluid.

2. 10. The method of claim 1, wherein the erectile dysfunction is caused by cardiovascular disease, diabetes, an anatomical defect, a nerve-related disorder, a hormone deficiency, a side effect of a medication, or any combination thereof.

3. 2. The method of claim 1, wherein the erectile dysfunction is neurogenic erectile dysfunction.

4. 4. The method of claim 3, wherein the neurogenic erectile dysfunction is caused by stroke, brain and / or spinal cord injury, diabetes, multiple sclerosis, Parkinson's disease, trauma from radical prostatectomy or radical pelvic surgery, or any combination thereof.

5. 10. The method of claim 1, wherein the erectile dysfunction comprises smooth muscle-related, arterial dilation, venous compression, or nerve atrophy.

6. A therapeutically effective amount of mesenchymal stem cells derived from the amniotic fluid is administered to improve smooth muscle-related, intracorporeal pressure, arterial dilation, venous compression, or neuronal atrophy in a dose of about 1×10 ６ ~2×10 ８ The method of claim 1, wherein the subject is administered a dose in the range of

7. The method of claim 1, wherein the mesenchymal stem cells are positive for CD73, CD90, CD105, nestin, Sox2, or any combination thereof, the mesenchymal stem cells are negative for CD34, CD45, CD14, CD11b, CD79α, CD19, HLA-DR, or any combination thereof, and the mesenchymal stem cells have a fibroblast-like morphology in adherent culture.

8. The method of claim 1 , wherein the mesenchymal stem cells have osteogenic potential, adipogenic potential, chondrogenic potential, or any combination thereof.

9. The mesenchymal stem cells are (a) obtaining an amniotic fluid sample from first, second or third trimester amniotic fluid by amniocentesis or Caesarean section; (b) centrifuging the amniotic fluid sample at 200 x g for at least 5 minutes and removing the supernatant; (c) culturing the cells in a medium that is an alpha modified minimal essential medium supplemented with about 1-5% human platelet lysate or about 10-20% fetal bovine serum or a commercially available mesenchymal stem cell medium; (d) removing non-adherent cells after 2-4 days of culture and allowing the adherent cells to grow as colonies for at least 7-14 days; The adherent cells were trypsinized and cultured at 1000-9000 cells / cm ２ and (e) passage and expanding the adherent cells at a seeding density of 0.1 to 0.5% by weight.

10. 2. The method of claim 1, wherein the alpha modified minimal essential medium is further supplemented with about 4 ng / ml basic fibroblast growth factor.

11. 1. A method for treating erectile dysfunction, comprising administering to a subject in need thereof a therapeutically effective amount of a mesenchymal stem cell derived secretome, wherein the mesenchymal stem cells are derived from amniotic fluid.

12. 12. The method of claim 11, wherein the erectile dysfunction is caused by cardiovascular disease, diabetes, an anatomical defect, a nerve-related disorder, a hormone deficiency, a side effect of a medication, or any combination thereof.

13. 12. The method of claim 11, wherein the erectile dysfunction is neurogenic erectile dysfunction.

14. 14. The method of claim 13, wherein the neurogenic erectile dysfunction is caused by stroke, brain and / or spinal cord injury, diabetes, multiple sclerosis, Parkinson's disease, trauma from radical prostatectomy or radical pelvic surgery, or any combination thereof.

15. 12. The method of claim 11, wherein the erectile dysfunction comprises smooth muscle-related, arterial dilation, venous compression, or neuronal atrophy.

16. The method of claim 11, wherein the therapeutically effective amount of the mesenchymal stem cell secretome is about 200 to 2000 μL.

17. The method of claim 11, wherein the mesenchymal stem cells are positive for CD73, CD90, CD105, nestin, Sox2, or any combination thereof, and the mesenchymal stem cells are negative for CD34, CD45, CD14, CD11b, CD79α, CD19, HLA-DR, or any combination thereof.

18. The method of claim 11 , wherein the mesenchymal stem cells have a fibroblast-like morphology in adherent culture.

19. The method of claim 11 , wherein the mesenchymal stem cells have osteogenic, adipogenic, chondrogenic potential, or a combination thereof.

20. The secretome of the mesenchymal stem cell is (a) culturing amniotic fluid stem cells in a basal medium for about 24 to 72 hours until about 80% confluence; (b) collecting the culture medium supernatant after centrifugation at about 300 x g for about 10 minutes to remove dead cells and debris; and (c) filtering the supernatant through a 0.22 μm filter and storing the secretome at about −20° C.

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