KR20160060593A - PHARMACEUTICAL COMPOSITIONS FOR Erectile Dysfunction COMPRISING Clonal Mesenchymal Stem Cells - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating erectile dysfunction, which comprises stem cells obtained through a subfractionation culturing method.
Clonal BMSCs using the SCM formed according to the present invention significantly improve the erectile function by increasing the content of the endothelial cells and spongy smooth muscle of the penis and increasing the content of nNOS and nerve fibers of the penis, It can be used as an erectile dysfunction treatment.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating erectile dysfunction, which comprises clonal mesenchymal stem cells,

The present invention relates to a pharmaceutical composition for preventing or treating erectile dysfunction, which comprises stem cells obtained through a subfractionation culturing method.

Despite the development of surgical techniques such as nerve-sparing techniques and robotic procedures, it is a common complication of radical prostatectomy (RP) for the treatment of prostate cancer. Erectile dysfunction occurs. Despite bilateral nerve preservation RP, partial cavernous nerve injury (CNI) or neurapraxia is inevitable because the spongy nerve is located close to the prostate capsule and is very fine. Because the regeneration of the nerves is a very slow process, the long-term denervation of the penis causes structural changes in the erectile tissue, including loss of endothelium and smooth muscle, deposition of extracellular matrix, and cavernosal fibrosis, Causing irreversible damage. Although oral PDE5 (phosphodiesterase-5) inhibitors are generally effective for the treatment of ED, ED male patients caused by RP are less effective in this treatment than general ED patients. Therefore, there is a need for new treatments that can fundamentally treat pathological processes in the corpus cavernosum tissue for the effective treatment of ED.

Recently, at pre-clinical levels, stem cell therapy has received attention for treatment of ED. Various kinds of stem cells, including embryonic stem cells, as well as adult stem cells isolated from bone marrow, adipose tissue, skeletal muscle and umbilical cord blood have been used for nerve, vascular endothelial cell or smooth muscle regeneration in ED animal models. Mesenchymal stem cells (MSCs) are adult stem cells, and bone marrow-derived stem cells (BMSCs) are the most widely studied in preclinical studies of ED studies. In a previous study of animal models of ED caused by diabetes, senescence, or CNI, IC infusion of BMSCs alone or genetically modified BMSCs promoted the regeneration of penile neural, vascular endothelial cells, or smooth muscle cells to enhance erectile function .

The best known method of isolating mesenchymal stem cells involves fractionation of mononuclear cells from donor tissue via centrifugation using adherent cells and removal of non-adherent flow cells. Most clinical studies in the field of ED have used centrifuged cells to separate mesenchymal stem cells and then used adherent cell culture techniques. However, recent evidence suggests that mesenchymal stem cells isolated using conventional gradient centrifugation methods are heterogeneous and contain different differentiation potentials, which can lead to unpredictable results, and thus clinical Suggesting that application may be very limited. To obtain a homogeneous subset of clonal MSCs, the inventors established a new protocol and named it subfractionation culturing method (SCM). This is for preparing single cell-derived clonal BMSCs from a relatively small amount of bone marrow aspirate and is described in U.S. Patent No. 7,781,211, issued on Aug. 24, 2010, Quot ;, which is incorporated herein by reference in its entirety.

The present inventors have isolated a uniform population of mouse clonal BMSCs using SCM and confirmed that they are effective in treating an erectile dysfunctional mouse model induced by bilateral cavernosal nerve injury, and accomplished the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating erectile dysfunction.

An aspect of the present invention is to provide a pharmaceutical composition for preventing or treating erectile dysfunction, which comprises stem cells obtained through a subfractionation culturing method.

The erectile dysfunction may be arteriovenous, venous, hormonal, neurogenic, and the erectile dysfunction may be caused by spongiform nerve injury or by diabetes.

In addition, the pharmaceutical composition may be intracavernously administered intraperitoneally or intraperitoneally.

The stem cells may not have a substantial effect on the sponge collagen content. In addition, the pharmaceutical composition can increase the content of the smooth muscle of the sea surface of the penis and increase the content of nNOS and nerve microfibrils in the penis.

The subfractionation culturing method may comprise the following steps:

(i) placing the cell sample in the first vessel without centrifugation so that the more dense cells are at the bottom and the less dense cells are at the supernatant;

(ii) transferring a supernatant containing cells of low density in the first vessel to the second vessel;

(iii) locating cells present in the supernatant such that the more dense cells are at the bottom and the less dense cells are at the supernatant;

(iv) repeating steps (ii) and (iii) three or more times;

(v) isolating a single colony of multi-lineage stem cells or progenitor cells from the supernatant;

(vi) transferring the cells from the colony to the growth medium and culturing the cells;

(vii) expanding the cells in 4 to 14 passages; And

(viii) obtaining a bank of multi-line stem cells or progenitor cells.

In addition, the cells of step (vii) may be expanded to 4 to 6 passages, and the cells may include frozen cells after 4 to 6 passages. The freezing may be frozen at -110 ° C to -150 ° C or in liquefied nitrogen. The cells may further include thawing and expanding the cells to obtain fresh cells.

In addition, the cells of step (vii) may be extended to 8 to 10 passages, and the cells may include cells after 8 to 10 passages. The freezing may be frozen at -110 ° C to -150 ° C or in liquefied nitrogen. The cells may further include thawing and expanding the cells to obtain fresh cells.

In addition, the cells of step (vii) may be expanded to 11 to 14, and the cells may include cells after 11 to 14 passages. The freezing may be frozen at -110 ° C to -150 ° C or in liquefied nitrogen. The cells may further include thawing and expanding the cells to obtain fresh cells.

The culture container may have a flat bottom and be coated with a cell adhesive. In addition, the cell adhesive may comprise a polymer of any charged amino acid, and the cell adhesive may be collagen, polylysine, polyarginine, polyaspartate, polyglutamate, or a combination thereof.

Clonal BMSCs using the SCM formed according to the present invention significantly improve the erectile function by increasing the content of the endothelial cells and spongy smooth muscle of the penis and increasing the content of nNOS and nerve fibers of the penis, It can be used as an erectile dysfunction treatment.

FIG. 1 is a view showing a process of separating mouse marrow-derived stem cells (BMSCs) from mouse clonally. The subfractionation culturing method is a method for isolating highly homogeneous mouse clonal BMSCs from bone marrow aspirate. BMSCs refers to bone marrow-derived stem cells.
Figure 2 shows isolation and characterization of clonal BMSCs.
(A) ex vivo FIG. 5 shows the morphology of clonal BMSCs in the culture. FIG.
(B) Lt; RTI ID = 0.0 &gt; in vitro < / RTI &gt; differentiation of the clonal BMSCs. After proper induction, cells were stained with oil red O (adipocyte differentiation), alizarin red S (osteogenic differentiation), or safranin O (cartilage differentiation) to confirm multilineage differentiation ability .
(C) cells were positive for positive mouse MSC markers while negative for hematopoietic / endothelial markers.
(D) In a mixed lymphocyte reaction, co-culture of clonal BMSCs inhibited proliferation of T cells. BMSCs represent bone marrow-derived stem cells, and MSCs represent mesenchymal stem cells.
Figure 3 illustrates that clonal BMSCs transplantation restores ICP (intracavernous pressure) caused by electrical stimulation of the spongy nerve.
(A) a single IP (intraperitoneal) injection of PBS (20 μL) or polyclonal ^{BMSCs (3 × 10 5 cells /} 20 μL) single IC (intracavernous) injection, and clonal ^{BMSCs (3 × 10 5 cells /} 20 μL) of (ICP) response in the sham operation group or CNI mice induced after 2 weeks. The interval of stimulation was indicated by a bar.
The ratio of mean maximum ICP and total ICP (area under curve) to mean (B, C) mean systolic blood pressure (MSBP) was calculated for each group. Each bar represents the mean value (± SE) of N = 6 animals in each group. * P < Stomach Surgery and BMSC (IC) Group. # P < PBS group † P <0.05 vs. PBS group. BMSCs refers to bone marrow-derived stem cells, and CNI refers to spongy nerve damage.
Figure 4 shows that transplantation of clonal BMSCs increases the endothelial cell content and increases the endothelial content of the sponge.
(A) Single intracavernous (IC) injection of PBS (20 μL) or clonal BMSCs (3 × 10 ⁵ cells / 20 μL) and single intraperitoneal injection of clonal BMSCs (3 × 10 ⁵ cells / 20 μL) PECAM-1 staining results of cavernosal tissues isolated from stomach surgery group or CNI mice 2 weeks after injection. Scale bar = 100 μm.
(B) Immunofluorescent staining of sponge with antibodies against PECAM-1 (red) and phosphohistone H3 (PH3; green) in each group.
(C) Quantitative analysis of endothelial cell content in the cavernosal tissue was performed using an image analyzer. Each bar represents the mean value (± SE) of N = 6 animals per group. * P < Stomach Surgery and BMSC (IC) Group. # P < PBS group. † P <0.05 vs. PBS group.
(D) high-power field (screen magnification x 400) PH3-immunoprophilic endothelial cells. Each bar represents the mean value (± SE) of N = 6 animals per group. * P < Stomach surgery, BMSC (IC), and BMSC (IP) groups. # P < PBS group. Bone marrow-derived stem cells (BMSCs) represent bone marrow-derived stem cells, cavernous nerve injuries (CNI), and high-power fields (HPF).
Figure 5 is a graph showing that transplantation of clonal BMSCs increases sponge smooth muscle content.
(A) Intraperitoneal injection of a single IC (intracavernous) injection of PBS (20 μL) or clonal BMSCs (3 × 10 ⁵ cells / 20 μL) and a single IP (3 × 10 ⁵ cells / 20 μL) of clonal BMSCs After 2 weeks, sponge tissue isolated from stomach surgery group or CNI mouse was stained with anti-PECAM-1 and smooth muscle α-actin. Scale bar = 100 μm.
(B) Quantitative analysis of smooth muscle content in cavernosal tissues was performed using an image analyzer. Each bar represents the mean value (± SE) of N = 6 animals per group. * P &lt; Stomach surgery group. # P &lt; PBS group. † P <0.05 vs. PBS group. Bone marrow-derived stem cells (BMSCs) represent bone marrow-derived stem cells and cavernous nerve injuries (CNI) represent spongy nerve damage.
Figure 6 is a set of figures that demonstrate that transplantation of clonal BMSCs does not affect sponge collagen content.
(A) Intraperitoneal injection of a single IC (intracavernous) injection of PBS (20 μL) or clonal BMSCs (3 × 10 ⁵ cells / 20 μL) and a single IP (3 × 10 ⁵ cells / 20 μL) of clonal BMSCs After 2 weeks, it was the MT (Masson trichrome) staining of cavernosal tissues isolated from the stomach group or CNI mouse. Scale bar = 200 μm.
(B) Quantitative analysis of collagen content in cavernosal tissues was performed using an image analyzer. Bone marrow-derived stem cells (BMSCs) represent bone marrow-derived stem cells and cavernous nerve injuries (CNI) represent spongy nerve damage.
Figure 7 discloses the effect of clonal BMSCs transplantation on the nNOS and neurofilament contents of the penis.
(A) Intraperitoneal injection of a single IC (intracavernous) injection of PBS (20 μL) or clonal BMSCs (3 × 10 ⁵ cells / 20 μL) and a single IP (3 × 10 ⁵ cells / 20 μL) of clonal BMSCs After 2 weeks, the results of anti - nNOS staining of penile tissues from the stomach group or CNI mouse. Scale bar = 25 μm.
(B) The result of anti - nerve microfibres staining of penile tissues in each group.
BMSCs (bone marrow-derived stem cells) represent bone marrow-derived stem cells, CC (corpus cavernosum) represents penile cavernosum, CNI (cavernous nerve injury) represents spongiform nerve injury, and DNB (dorsal nerve bundle) represents dendritic bundles.
8 is a view showing the erectile function restoration of the clonal BMSCs transplantation in a diabetic erectile dysfunction mouse.
(A) Results of intracavernous pressure measurements of the sponge venous pressure after stimulation (5 V, 12 Hz, 1 ms) of spongy nerve in normal control or diabetic mice 2 weeks after treatment.
(B) the maximum ICP (intracavernous pressure) for MSBP (mean systolic blood pressure).
(C) the ratio of total ICP to MSBP (mean systolic blood pressure).
FIG. 9 shows the increase in the amount of sponge endothelial cells and smooth muscle cells through the transplantation of clonal BMSCs in a diabetic erectile dysfunction mouse.
Immunohistochemical staining of sponge tissue using antibodies against PECAM-1 was performed on normal control and diabetic mice two weeks after treatment. Significantly lower levels of spongy endothelial cells were observed in diabetic and PBS-treated diabetic mice than in normal control mice. IC injection of clonal BMSCs significantly restored sponge endothelial cell content (FIGS. 9A and 9B).
Two weeks after treatment, immunohistochemical staining of cavernosal tissues was performed using antibodies against smooth muscle alpha -actin in normal control and diabetic mice. Significantly lower levels of sponge smooth muscle cells were observed in diabetic and PBS-treated diabetic mice compared to normal control mice. IC injection of clonal BMSCs in diabetic mice restored sponge smooth muscle content (FIGS. 9A and 9C).
10 is a graph showing the results of induction of phosphorylation of endothelium-derived nitric oxide synthase by transplantation of clonal BMSCs in a diabetic erectile dysfunction mouse. IC injection of BMSCs restored endothelial cell nitric oxide synthase phosphorylation to normal control level.
Figure 11 shows the inhibitory effect of sponge fibrosis through transplantation of clonal BMSCs in diabetic erectile dysfunction mice. IC injection of clonal BMSCs inhibited sponge fibrosis in diabetic mice and there was no difference in the level of sponge collagen.
Figure 12 is an elevation of penile nNOS and neuronal fine fiber content through clonal BMSCs transplantation in diabetic erectile dysfunction mice. IC injection of clonal BMSCs in diabetic mice significantly restored neuronal microfibrils and nNOS content.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating erectile dysfunction, which comprises stem cells obtained through a subfractionation culturing method.

Hereinafter, the present invention will be described in detail.

As used herein, "body sample" means any sample obtained from a mammal in which a single type of cell is to be isolated. Such body samples include bone marrow samples, peripheral blood, cord blood, fatty tissue samples, and cytokine-activated peripheral blood.

As used herein, "mammal" or "subject" for discussing the source and treatment of a cell include humans, domestic and farm animals, and zoo, sports, or pets such as dogs, cats, , Pigs, rats, mice, rabbits, and the like. Preferably, the mammal is a human.

As used herein, "cell sample" means any sample comprising a mixture of different types of cells, including bone marrow samples, peripheral blood, cord blood, fatty tissue samples and cytokine-activated peripheral blood.

As used herein, a "homogeneous" population generally indicates that cells of the same type are present in the population. By "substantially homogeneous" is meant about 80% homogeneity or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

As used herein, "low density cells" refers to cells that are less dense than others in the sample and that are the subject of sequestration. Low-density cells include, but are not limited to, multi-lineage stem cells, progenitor cells, and other bone marrow stromal cells.

As used herein, "MLSC" means multi-line stem cells.

As used herein, "MLSC / PC" means a multi-stem cell or progenitor cell.

As used herein, "MSC" means bone marrow stromal cells or mesenchymal stem cells.

The erectile dysfunction may be of the arteriovenous, venous, hormonal, neurogenic type, and the erectile dysfunction may be caused by spongiform nerve injury. The erectile dysfunction may also be diabetic erectile dysfunction.

In addition, the pharmaceutical composition may be intracavernously administered intraperitoneally or intraperitoneally.

The stem cells may not have a substantial effect on the sponge collagen content. In addition, the pharmaceutical composition can increase the content of the smooth muscle of the sea surface of the penis and increase the content of nNOS and nerve microfibrils in the penis.

The subfractionation culturing method may comprise the following steps:

(i) placing the cell sample in the first vessel without centrifugation so that the more dense cells are at the bottom and the less dense cells are at the supernatant;

(ii) transferring a supernatant containing cells of low density in the first vessel to the second vessel;

(iii) locating cells present in the supernatant such that the more dense cells are at the bottom and the less dense cells are at the supernatant;

(iv) repeating steps (ii) and (iii) three or more times;

(v) isolating a single colony of multi-lineage stem cells or progenitor cells from the supernatant;

(vi) transferring the cells from the colony to the growth medium and culturing the cells;

(vii) expanding the cells in 4 to 14 passages; And

(viii) obtaining a bank of multi-line stem cells or progenitor cells.

In addition, the cells of step (vii) may be expanded to 4 to 6 passages, and the cells may include frozen cells after 4 to 6 passages. The freezing may be frozen at -110 ° C to -150 ° C or in liquefied nitrogen. The cells may further include thawing and expanding the cells to obtain fresh cells.

In addition, the cells of step (vii) may be extended to 8 to 10 passages, and the cells may include cells after 8 to 10 passages. The freezing may be frozen at -110 ° C to -150 ° C or in liquefied nitrogen. The cells may further include thawing and expanding the cells to obtain fresh cells.

In addition, the cells of step (vii) may be expanded to 11 to 14, and the cells may include cells after 11 to 14 passages. The freezing may be frozen at -110 ° C to -150 ° C or in liquefied nitrogen. The cells may further include thawing and expanding the cells to obtain fresh cells.

The culture container may have a flat bottom and be coated with a cell adhesive. In addition, the cell adhesive may comprise a polymer of any charged amino acid, and the cell adhesive may be collagen, polylysine, polyarginine, polyaspartate, polyglutamate, or a combination thereof.

The pharmaceutical composition of the present invention can be formulated by a method known to a person skilled in the art. For example, it can be used parenterally in the form of an aseptic solution of water or other pharmaceutically acceptable liquid, or an injectable suspension, if necessary. For example, a pharmaceutically acceptable carrier or medium is suitably combined with sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, It is conceivable to formulate the composition by admixing it in a unit dosage form required for the accepted pharmaceutical practice. The amount of the active ingredient in the preparation is intended to be such that an appropriate amount of the indicated range can be obtained. In addition, the sterile composition for injection may be formulated according to the conventional formulation by using a liquid such as distilled water for injection.

Examples of the aqueous solution for injection include physiological saline, isotonic solutions containing glucose or other auxiliary drugs such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, For example, ethanol, polyalcohols such as propylene glycol, polyethylene glycol, and nonionic surfactants such as polysorbate 80 (TM) and HCO-50 can be used in combination.

Examples of the oily solution include sesame oil and soybean oil, and can be used in combination with benzyl benzoate and benzyl alcohol as solubility aids. In addition, it can be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, an anhydrous agent such as hydrochloric acid, a stabilizer such as benzyl alcohol, phenol and an antioxidant. The prepared injections are usually filled into a suitable ampoule.

The administration to the body of the patient is preferably parenteral administration. Specifically, the administration is one to three times in a vein, but the administration may be more. The administration time may be a continuous administration for a short time or for a long time. More specifically, examples include injection form, transdermal dosage form, and the like. Examples of the injectable form include, for example, intravenous injection, intraarterial injection, selective intraarterial injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intracerebral injection, intracerebral injection, intramedullary injection, Intravenous injection or intraperitoneal injection, although it can be administered. In the case of intravenous injection, since transplantation is possible in the general transfusion procedure, there is no need to perform surgery on the patient and further local anesthesia is not required, so that the burden on both the patient and the physician is light. It is also very suitable as a point of operation in the ward. In consideration of the development of future emergency care, it is possible to consider administration during the emergency return or on the spot of the onset.

In order to treat erectile dysfunction, the clonal BMSCs of the present invention can be administered to a patient in a therapeutically effective amount. The therapeutically effective amount is not particularly limited, but is preferably 1 x 10 ⁴ cells / kg to 1 x 10 ⁸ cells / kg, more preferably from 1 × 10 ⁵ cells / kg to 1 × 10 ⁷ cells / kg, and most preferably from 5 × 10 ⁵ cells / kg to 5 × 10 ⁶ cells / kg. The above method of administration is not particularly limited, but any of the parenteral administration methods can be used, and systemic administration or local administration is possible, but systemic administration is more preferable, and intravenous administration is most preferable.

Layer separation  Culture technology

In the present invention, a method called the subfractionation culturing method utilizes a method of separating a very homogeneous population of multi-system stem cells (MLSC) from a source such as a body sample or human bone marrow. A total of 16 bone marrow cell lines were established from 1 ml bone marrow aspirate. Among 16, four cell lines expressing different phenotypes were characterized by FACS analysis. All of these cell lines showed features of multi - line stem cells such as self - renewal function and differentiation potential into mesodermal, ectodermal, endodermal lineage cells.

The bone marrow MSC is known to be difficult to separate without contamination by hematopoietic cells. In order to apply to clinical settings, it is important to have a uniform population of MSCs to solve the immune source problem and accurately assess the clinical effect. Traditionally, separation of a uniform population of MSCs was performed by MSC-specific antibody column purification. However, this method is not sufficient because such a complete MSC-specific antibody is still not possible.

The principle of the present invention for isolating MLSCs from biological samples, such as bone marrow samples, is that multi-line stem cells or progenitor cells have low cell densities and thus can be separated from other cells in the sample accordingly. For example, mature MSCs are larger than rapid self-renewing (RS) cells. RS cells are known to have greater capacity for multiple lineage differentiation.

In another embodiment, collagen or polylysine-coated culture dishes were used to obtain more adherent stem cells. Applicants have found that a positively charged cultured surface with positive or negative stimulates adhesion to the surface of stem cells compared to uncoated dish surfaces. Approximately two to three times more cells were attached to the collagen or polylysine-coated culture dish than the uncoated dish, respectively (data not shown). Similar results were obtained in other human bone marrow cell populations and in three different lineages of mouse bone marrow samples (data not shown), with respect to the yield of single cell-derived bone marrow cell colonies, indicating that this protocol is compatible with other bone marrow- Consistent and can be applied to the separation of MLSC in other species.

Thus, in one embodiment, the bottom of the culture dish comprises a positively charged amino acid such as polylysine, polyarginine, or a negatively charged amino acid such as polyaspartate, polyglutamate, May be coated by a combination to help the stem cells or progenitor cells adhere to the bottom of the dish.

In order to carry out the layer separation culture method of the present invention, any type of cells such as erythrocytes or leukocytes can be removed from the sample in advance, since most of the heavier or denser cells can be removed in the first two 2 hour incubation steps There is no need to use any type of centrifugation to remove. At this point, it is not necessary to pre-treat the cells with any enzyme that digests any material between the cells. Thus, one of the advantages of the system of the present invention is that it can be used as a commonly used density-gradient capable of introducing pollutants such as Picoll, Ficoll or Ficoll-hypaque into cell culture fluids Centrifugation and mononuclear cell fractionation steps. Thus, the layer separation culture method of the present invention is a simple, effective and economical protocol for separating highly homogeneous MLSCs from body samples, preferably bone marrow samples.

Alternatively, conventional density-gradient centrifugation separate / fractioned mononuclear cells of MSC separation can be introduced into dish D1 to obtain single cell-derived colonies and isolate a uniform population of stem cells or progenitor cells have. Therefore, in the fractionation culture method, mononuclear cells fractionated by conventional density-gradient centrifugation methods can be used.

The present application describes a variety of characteristics in cell surface protein expression of single cell-derived stem cell lines, indicating that there are several types of stem cells or progenitor cells present in a biological sample, particularly the exemplified bone marrow sample. The isolated MLSCs were generally negative for CD34, HLA-DR, CD73, CD31, CD166, HLA class I, or slightly positive and very positive for CD44, CD29, CD105. However, some cell lines from the plates of D4 and D5 show a clear level of surface protein, indicating that some other multi-lineage stem cells or progenitor cells may be present in the bone marrow. Hung et al. Assumed that bone marrow could include many other groups of MSCs in surface marker assays [24]. These MSCs with a variety of surface markers can represent different cell differentiation potentials. Therefore, due to the isolation of single cell-derived homogeneous stem cells by the layer separation culture method of the present invention, the cells of this group are present in bone marrow or other specially isolated body samples, As long as the ability does not change, it becomes possible to separate tissue-specific stem cells or related progenitor cells. The safety and efficacy of MSC therapy and cell transplantation treatment is enhanced by being able to characterize cell sub-populations with specific properties, as provided herein.

The present application presents a novel method of separating a highly homogeneous population of MLSC lines from a single cell from any other body sample, particularly bone marrow, with multiple lineage differentiation potential into regenerative function and ectodermal, mesodermal and endodermal lineage cells . By eliminating the density-gradient centrifugation and mononuclear cell fractionation steps without the need to use antibodies to isolate stem cells, the sub-fractionation method of the present invention is simple, efficient and economical, A cluster of one MLSC is created.

Induction, differentiation / A transformant

Inducing, differentiating / transforming agents for the endoderm cell lineage can be used for the type of cells such as liver, lung, pancreas, thyroid and intestinal cells, hepatocyte growth factor, oncostatin-M, Fibroblast growth factor-4, basic fibroblast growth factor, insulin, transferrin, selenius acid, BSA, linolenic acid, ascorbate 2-phosphate, VEGF and dexamethasone.

Induction, differentiation / A transformant

The induction, differentiation / transformation agent for the mesodermal cell line is selected from the group consisting of insulin, transferrin, selenic acid, BSA, linolenic acid, TGF-beta1 , TGF-? 3, ascorbate 2-phosphate, dexamethasone,? -Glycerophosphate, ascorbate 2-phosphate, BMP and indomethacin.

Induction, differentiation / A transformant

The induction, differentiation / transformation agent for the ectodermal cell line can be used for dibutyrylcyclin AMP, isobutylmethyl xanthine, human epithelial growth factor, basic &lt; RTI ID = 0.0 &gt; Fibroblast growth factor-8, brain-induced neurotrophic factor, and / or other neurotrophic factor.

Layer separation  Treatment of Erectile Dysfunction with Cells Obtained by Culture Method

Recently, stem cell therapy has attracted attention, and BMSCs (bone marrow-derived stem cells) are one of the mesenchymal stem cells that have been studied in the gut of the stem cells used in erectile dysfunction. However, the biggest obstacle to the clinical application of stem cell therapies is the non-uniform nature of the isolated cells, which results in different therapeutic outcomes.

Accordingly, the present inventors confirmed the effect of the mouse bronchial BMSCs obtained from a single colony using a subfractionation culturing method (SCM) on erectile dysfunction in a cavernous nerve injury (CNI) mouse model.

20 week old C57BL / 6J mice were divided into 4 groups: bilateral CNI groups treated with a single IC (intracavernous) injection of the stomach group, PBS (20 μL) or clonal BMSCs (3 × 10 ⁵ cells / 20 μL) Clonal BMSCs (3 × 10 ⁵ cells / 20 μL) were injected into a single IP (intraperitoneal injection) bilateral CNI group.

The clonal BMSC line was analyzed for cell-surface epitope and differentiation potential using fluorescence-activated cell sorting. Two weeks after bilateral spinal nerve injury and treatment, erectile function was measured by electrical stimulation of the spongy nerve. The penis was retrieved for histological analysis.

Clonal BMSCs expressed cell surface markers of mesenchymal stem cells and were capable of differentiating into various lines including adipocytes, osteogenic cells, and cartilage cells. In ICI and IP injection of both clonal BMSCs, both the sponge endothelium and smooth muscle content, and the content of nerve nerve fibers and nerve fibers in the CNI mice were recovered to low levels. IC injection of clonal BMSCs significantly restored erectile capacity, reaching 90-100% of the stomach control value, whereas IP injection of clonal BMSCs partially restored erectile capacity. The infusion can be intravenous in the cavernous vein.

The present inventors have established a uniform population of mouse clonal BMSCs using SCM; Clonal BMSCs successfully restored the erectile capacity of CNI mice. The uniform nature of clonal mesenchymal stem cells makes this clinical application possible.

The present inventors suggest that IC or IP injection of clonal BMSCs significantly restores spongy endothelial and smooth muscle cell content, and penile nNOS and nerve fiber content in CNI mice. IC infusion of clonal BMSCs significantly restored erectile capacity, reaching 90-100% of the stomach control value, whereas IP infusion of clonal BMSCs partially restored erectile capacity.

In the present invention, we used a mouse clonal BMSCs strain isolated from a relatively small amount of bone marrow aspirate using a subfractionation culturing method (SCM); Clonal BMSCs are characterized by cell phenotype and differentiation potential. Clonal BMSCs expressed cell surface markers of MSCs and differentiated into various cell lines including adipose, ovule, and cartilaginous cells. In the past, the present inventors established six human BMSC lines using SCM, and confirmed that the established clonal BMSC line was very diverse in cell surface epitope, differentiation potential and cytokine secretion, Of stem cells are present. Through these conventional studies, the present inventors have suggested that different cell types exist in a uniform subpopulation of clonal BMSCs, which may have therapeutic advantages in various clinical situations. The advantages of the protocol of the present invention for establishing clonal stem cell lineage are as follows. First, only a small amount of bone marrow aspirate is needed. Second, the protocol of the present invention eliminates the possibility of contamination such as Picoll, Ficoll-hypaque, which does not require a gradient-centrifugation step and thus can be harmful to stem cells. Third, no enzymatic treatment or filtering procedure is required to isolate stem cells. Finally, we obtained a very uniform population of mesenchymal stem cells. A major limitation to the clinical application of stem cell therapies is the heterogeneity of isolated stem cells using conventional methods. A uniform population of clonal MSCs obtained from single cell-derived colonies can avoid this problem and facilitate clinical application of stem cell therapies.

Previous studies have shown that IC infusion of BMSCs in diabetic rats significantly increases spongy endothelial and smooth muscle content. Similar to these results, IC injection of mouse clonally BMSCs in CNI mice induced complete recovery of spongy endothelial and smooth muscle content. In animal models, CNI has been shown to induce increased expression of the profibrotic factor and induce cavernous fibrosis. Increased protein expression of collagen I and III and increased sponge collagen content were observed in the cavernosal tissues of the male CNI mice and RP. However, in the present invention, it was confirmed that the sea surface collagen content determined using Masson's trichrome staining did not increase after CNI. These results can be attributed to relatively short-term CNI. The balance between the spongy smooth muscle and connective tissue is crucial to maintaining the veno-occlusive mechanism. If the spongy smooth muscle content is less than the critical amount, the vein-occlusion mechanism does not work inevitably. In the present invention, based on the ratio of collagen to smooth muscle in CNI mice, the IC and IP injection of mouse clonal BMSCs significantly reduced spongiform fibrosis, mainly due to restoration of smooth muscle content, Of the total.

It has been reported that BMSCs can differentiate into spongy endothelial cells and smooth muscle cells, but accumulated evidence suggests that the near-paracrine activity of MSCs plays an important role in providing stem cell therapy benefits. This concept was further supported by experiments with MSCs conditioned medium or lysates. Previous studies in diabetic rats have demonstrated that IC infusion of BMSC-conditioned medium significantly restored erectile function, but not to the extent observed in the BMSC-treated group. The results of the BMSC-conditioned medium under ex vivo conditions provide indirect evidence of the paracrine activity of stem cells and may be used to identify serum proteins and immune systems that may influence nutrient or secretory activity of BMSCs And the like, could not fully reflect the in vivo situation.

In the present invention, BMSCs IP was administered to obtain direct evidence of such basophil secretory activity. Although no obvious recovery of erectile function as observed in the IC injection group was observed, IP injection of BMSCs also significantly restored erectile function in CNI mice by restoring spongy endothelial and smooth muscle mass, It supports the paracrine effect.

The number of intact nNOS-positive neurons is important in maintaining erectile function. In the present invention, in CNI mice, IC and IP injection of clonal BMSCs significantly restored the nNOS content of the penis.

The new protocol of the present invention was used to establish a human clonal MSC library with various differentiation potentials and cytokine secretion profiles. Such a human clonal MSC library will enable cause-specific or individualized stem cell treatment for erectile dysfunction due to a variety of causes. For example, for the combined neurogenic and vasculogenic erectile dysfunction resulting from RP or prolonged diabetes mellitus for the treatment of prostate cancer, a high fractional profile of neurotrophic and angiogenic factors, indicating high potential for nerve and vascular regeneration Lt; RTI ID = 0.0 &gt; MSC &lt; / RTI &gt;

This invention is the first study showing that clonal BMSCs obtained from single cell-derived colonies are useful for the treatment of ED. In the present invention, the inventors used SCM to isolate and identify a uniform population of mouse clonal BMSCs. In vitro, clonal BMSCs can differentiate into a variety of cell lines, including adipocytes, osteocytes, and cartilage cells. Both IC and IP injection of clonal BMSCs successfully restored erectile function in CNI rats by restoring the endothelial cells, smooth muscle, and neuronal cell aggregates. A major limitation when applying stem cells to clinical trials is the heterogeneous heterogeneity of isolated cells. The obtaining of homogeneous clonal MSCs from single cell-derived colonies can accelerate clinical applications.

The invention is not to be limited in scope by the specific embodiments described herein. Indeed, in addition to the description herein, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such variations are within the scope of the appended claims. The following examples serve to illustrate the present invention and are not intended to be limiting.

Example

Example  One- Nerve toughness  How to check the effectiveness of erectile dysfunction treatment

Example 1 .One - Clonal BMSC  Separation and construction of systems

Using 5-ml culture medium consisting of DMEM (Dulbecco's modified Eagle's Medium) -low glucose (GIBCO-BRL, Life Technologies, Gaithersburg, MD, USA), 10% fetal bovine serum, and 1% penicillin / streptomycin , And bone marrow samples were collected from the tibia and femur of 5-week-old C3H mice by washing the bone marrow cavity. Next, 15 ml of complete growth medium was added to the mixture. The mixture was cultured in a 100-mm culture dish. After incubation for 1 hour at 50 ml / L CO ₂ and 37 ° C, the supernatant was transferred to a clean medium 100-mm dish (Fig. 1). After the second one-hour incubation, the supernatant was transferred from the intermediate dish to a new dish (D1) and incubated for an additional 1 hour. Then, the supernatant was transferred from D1 to a new dish (D2), cultured for 1 day, transferred to a new dish (D3), and cultured for 1 day. This procedure was repeated two times by culturing for 1 day and 2 days (D4 and D5, respectively). After 7 to 14 days of culture, well-separated, single-cell-derived colonies on the culture plate were removed after 2-3 min of treatment with trypsin / EDTA (ethylene diamine tetraacetic acid) in a cloning cylinder (GIBCO-BRL) And separated. The culture was transferred to a 6-well plate and transferred to a large culture flask where cells could continue to expand. Once the cells reach 70-80% confluence, they are recovered using trypsin / EDTA and then treated with 50-100 cells / cm ² . 5 mouse bone marrow samples were used to obtain 10 and 20 colony on D1 to D2 dishes and a number of colonal BMSC lines were established and one (D101) was selected for use in the present invention.

Example  1.2 - Immunocytochemistry and flow cytometry

For cell surface antigen phenotypes, clonal BMSCs of 5-7 passages established in 175 cm ² flasks were treated with trypsin / EDTA and washed twice with PBS. Cells were incubated with FITC-conjugated antibodies for 30 min at 4 ° C and then washed twice with PBS containing 0.1% BSA. The following antibodies were used in flow cytometry: CD34 (Serotec, Oxford, UK), CD44 (Serotec), CD45 (BD Biosciences Pharmingen, San Diego, CA, USA), CD103 (BD Biosciences Pharmingen), CD105 (BD Biosciences Pharmingen), MHC Class II (BD Biosciences Pharmingen), and Sca-1 (BD Biosciences Pharmingen). Cells were washed twice with PBS containing 0.1% BSA and analyzed by flow cytometry of a 525 nm filter against green FITC fluorescence.

Example  1.3 - In vitro  differentiation

To compare the differentiation potentials, the following methods were used to induce clonal BMSCs to differentiate into adipocytes, osteocytes, or chondrocytes.

For cartilage differentiation, a pellet culture system was used. Approximately 2 × 10 ⁵ clonal BMSCs were placed in a 15-mL polypropylene tube (Falcon, Bedford, Mass., USA) and centrifuged. The cell pellet was resuspended in 10 ng / mL TGF- beta 1 (R & D Systems), 10 ng / mL TGF- beta 3 (R & D Systems), 50 μg / mL ascorbic acid (Sigma Chemical Co., St Louis, (6.25 ug / mL transferrin, 6.25 ng / mL selenic acid, 1.25 mg / mL BSA, and 1 mg / mL dexamethasone (Sigma), 40 ug / mL proline L of CO ₂ supplemented with α-MEM supplemented with 0.25 mg / mL linoleic acid (Becton Dickinson, Bedford, Mass., USA) at 37 ° C and 50 mL / L CO ₂ . The cartilage differentiation medium was changed every 3 days for 3 weeks. For microscopic observation, pellets were embedded in OCT compound (Sakura Finetek, Torrance, Calif., USA), frozen in 8 μm sections and stained with toluidine blue or sapranin O.

To investigate osteoblast and adipocyte differentiation, a monolayer culture system was used. For osteogenic differentiation, the clonal BMSCs were plated in 12-well plates at 50 cells / cm ^2, and cultured until the aggregate (confluence). The cultures were incubated with 10% FBS, 50 ug / mL ascorbic acid (Sigma), 10-8 mol / L dexamethasone (Sigma), 10 mmol / L? -? - glycerophosphate (Sigma), and 1 mmol / L dibutyryl- and cultured in osteogenic differentiation medium consisting of alpha-MEM supplemented with cyclic AMP (db-cAMP). db-cAMP was added only during the first 4 days. The medium was changed every 3 days for 3 weeks. Cells were fixed in 10% formalin (Sigma) for 10 min at room temperature and stained with 0.1% Alizarin Red S (Sigma).

In order to evaluate the fat cell differentiation, clonal BMSCs were cultured in α-MEM (20% FBS) in 12-well plates until the plating, and to form an aggregate with 50 cells / cm ^2. The cultures were then incubated with 10% fetal bovine serum, 10-7 mol / L dexamethasone (Sigma), 10 μg / mL insulin (Sigma), 0.5 μmol / L IBMX (1-methyl-3-isobutylxanthine) And cultured in adipocyte differentiation medium consisting of DMEM-high glucose supplemented with 50 μg / mL indomethacin (Sigma). The cultures were incubated for 4 days at 37 ° C and 50 mL / L CO ₂ . Cells were fixed with 10% formalin (Sigma) for 10 minutes at room temperature and stained with Oil-Red-O for 10 minutes.

Example  1.4 - Nerve toughness  Erectile dysfunction induction and treatment of animals

12 week old C57BL / 6J mice were used in the present invention. The experiment was approved by the University's Animal Care and Use Committee. Mice were divided into 4 groups: bilateral CNI groups treated with a single IC (intracavernous) injection, and clonal BMSCs (3x10 ⁵ cells in 20 μL of PBS) in PBS (20 μL) or cBMSCs 10 ⁵ cells / 20 μL of PBS) were injected intraperitoneally.

The stomach group was exposed to the prostate so that bilateral spongy nerves could be visualized without direct manipulation of the spongy nerve. In the CNI group, spongy nerve (Karl Stortz Co., Tuttlingen, Germany) was used instead of jaws. Hemostasis was applied to full span closure of each spongy nerve 1 mm from the ganglion for 2 minutes. After CNI, the penis was exposed using a sterile technique. Clonal BMSCs were administered to the central portion of the cavernosum using a 30-gauge syringe. The incision was closed using a 6-O Vicryl (polyglactin 910) suture. All procedures were performed with the help of a dissecting microscope (Zeiss, Gottingen, Germany). We evaluated the erectile function by electrical stimulation of the spongy nerve (N = 6 per group) after recovery of the penis for histological examination and 2 weeks after treatment with CNI.

Example  1.5 - Erectile function measurement

The erectile function was measured by electrical stimulation of the spongy nerve as described above.

Example  1.6 - Histological examination

Prior to paraffin embedding, the medial portion of each penis piece was immediately fixed in 10% formalin in PBS. The specimens were cut with 4-μm sections and stained with Masson trichrome.

For fluorescence microscopy, the penile tissue was fixed in 4% paraformaldehyde at 4 ° C for 24 hours, and then frozen tissue sections (12 μm thick) were applied to PECAM-1 (platelet / endothelial cell adhesion molecule, endothelial marker; Antibody to smooth muscle alpha -actin (smooth muscle cell marker, anti-inflammatory cytokine, anti-inflammatory cytokine, Sigma-Aldrich (Sigma-Aldrich; 1: 100), or neuronal nitric oxide synthase (Santa Cruz Biotechnology, Santa Cruz, CA, USA; And incubated overnight at 4 ° C. Control sections were incubated without primary antibody. After washing several times with PBS, the sections were incubated with tetramethyl rhodamine isothiocyanate- or FITC-conjugated secondary antibody for 2 hours at room temperature. Samples were stained for nuclear staining by storing them in a solution containing DAPI (4,6-diamidino-2-phenylindole, Vector Laboratories, Inc. Burlingame, Calif., USA). The signals were visualized and digital images were obtained using a confocal microscope (FV1000, Olympus, Tokyo, Japan).

The endothelial cells, smooth muscle cells and collagen content of the sponges were analyzed using a quantitative image analysis system (National Institutes of Health [NIH] Image J 1.34, http://rsb.info.nih.gov/ij/index.html ) The number of phospho-histone H3-immunopositive endothelial cells was counted at a screen magnification of x400 at 6 or 8 different positions. The values are displayed for each high power field.

Example  1.7 - Statistical analysis

Results are expressed as mean ± standard error. Group comparisons of parametric data were made by one-way ANOVA according to the Newman-Keuls post hoc test. We obtained non-parametric data using the Kruskal-Wallis test. Statistical analysis was performed using SigmaStat 3.5 software (Systat Software Inc., Richmond, Calif.). When P was less than 5%, it was judged to be significant.

Example  2- Nerve toughness  Efficacy of erectile dysfunction treatment

Example  2.1 - Clonal Of BMSCs  Separation and characterization

To obtain highly homogenous mesenchymal stem cells, the present inventors isolated clonal BMSCs from bone marrow aspirates from C3H mice as previously described. Cells used in this study showed typical fibroblast morphology in conventional two-dimensional cell culture (Figure 2A). To determine if the isolated cells were mesenchymal stem cells, differentiation potentials and expression of cell surface markers were examined. The cells showed excellent multilineage differentiation potential. When in vitro cells were induced using adipocytes, osteoblast, or cartilage cell culture media, cells were cultured in a typical adipocyte (oil red O stain), osteoblast (alizarin red S stain) and osteoblast (sopranin O Staining) (Fig. 2B). To analyze the expression of MSC cell surface markers, flow cytometry using several antibodies was used. Cells expressed the known MSC markers CD44 and Sca-1, but the hematopoietic and endothelial markers CD34, CD45, and MHC Class II were not expressed (FIG. 2C). To test the immunosuppressive potential of clonal BMSCs, mixed lymphocyte reaction assays were performed. Inhibition of T cell proliferation was measured using antibody-activated splenocytes in the presence of clonal BMSCs. As shown in Figure 2D, the clonal BMSCs showed a T-cell inhibition potential even when the BMSCs to splenocyte ratio was 1: 100. These results show that clonal BMSCs isolated from mouse BM have MSC characteristics.

Example  2.2 - CNI  In mice, Clonal BMSCs  Confirmation of erectile function restoration of transplant

After 2 weeks of treatment, intravenous tracing was performed in the stomach control or CNI mice after stimulation of the spongy nerve (5 V, 12 Hz, 1 ms) for 1 minute and the result is shown in FIG. 3A .

The maximum ICP and total ICP for mean systolic blood pressure (MSBP) were significantly lower in PBS-treated CNI mice than in the control group.

Single IC infusion of clonal BMSCs induced recovery of erectile capacity compared to PBS-treated CNI mice, reaching 90-100% of the control value, whereas single IP injection of clonal BMSCs partially restored erectile capacity (FIGS. 3B and 3C). No detectable difference in systemic blood pressure was observed between the four experimental groups.

Example  2.3 - CNI  In mice, Clonal BMSCs  Transplant Endothelial cell  Induce proliferation Spongy through  Increase in endothelial content

Immunohistochemical staining of sponge tissue with antibodies against PECAM-1 was performed on the stomach control and CNI mice 2 weeks after treatment. Significantly lower levels of spongy endothelial cells were observed in PBS-treated CNI mice than in the control mice above. Both IC and IP scans of clonal BMSCs significantly restored sponge endothelial content (Figs. 4A and 4C). Immunostaining was used to determine the number of endothelial cells positive for phosphohistone H3 (nucleoprotein expressing cell proliferation) in order to investigate whether clonal BMSCs induce spongy endothelial cell proliferation through endothelial cell proliferation. CNI mice injected with IC or IP with clonal BMSCs showed significantly more phosphohistone H3-positive endothelial cells in cavernous sinusoids than in the control or PBS-treated CNI mice. In contrast, little or no phosphohistone H3-positive endothelial cells were observed in the stomach control or PBS-treated CNI mice (FIGS. 4B and 4D).

Example  2.4- CNI  In mice, Clonal BMSCs  Increased smooth muscle content through transplantation

Two weeks after treatment, immunohistochemical staining of cavernosal tissues was performed using antibodies against smooth muscle alpha -actin in the control and CNI mice. Significantly lower levels of sponge smooth muscle cells were observed in PBS-treated CNI mice compared to the control group Baus. Both IC and IP infusion of clonal BMSCs in CNI mice restored sponge smooth muscle content (Figure 5). Masson trichrome staining was also used to determine the sponge collagen content. No difference in detectable sponge collagen content was observed between the four experimental groups (FIG. 6). In corpus cavernosum, the degree of cavernous fibrosis determined based on the ratio of collagen to smooth muscle content was significantly higher in PBS-treated CNI mice than in the control group. Both IC and IP injection of clonal BMSCs significantly reduced cavernosal fibrosis in CNI mice.

Example  2.5 - CNI  In rats Clonal BMSCs  Penis through transplantation nNO  And Nerve microfiber  Increase in content

The expression of nNOS and nerve fibers in the cavernosal tissues or spinal cord bundles was significantly lower in the PBS-treated CNI group than in the control group. IC injection of clonal BMSCs in CNI mice significantly restored neuronal fine fibers and nNOS content. Expression of nNO and nerve fiber content increased after IP injection of clonal BMSCs, but not at the level of the IC injection group (Fig. 7).

Example  3- Blood vessel toughness  How to check the effectiveness of erectile dysfunction treatment

Example  3.1 - Induction and treatment of animal models

16 weeks old C57BL / 6J mice were used in the present invention. The experiment was approved by the University's Animal Care and Use Committee. Mice were divided into 4 groups: diabetic group treated with single IC (intracavernous) injection in normal control, diabetic group, PBS (20 μL) or cBMSCs (3 × 10 ⁵ cells in 20 μL of PBS). PBS or clonal BMSCs were administered to the central portion of the cavernosum using a 30-gauge syringe.

Diabetes mellitus was induced in 8-week-old C57BL / 6J mice by intraperitoneal injection of streptozotocin (Sigma, USA) in PBS for 5 consecutive days at a dose of 50 mg / kg body weight per mouse Respectively. After 8 weeks from the last injection of streptozotocin, blood glucose was measured, and mice having a pre-meal blood glucose level of 250 mg / dL or more and a postprandial glucose level of 500 mg / dL were selected and used in the present invention.

Example  3.2 - Erectile function measurement

The erectile function was measured by electrical stimulation of the spongy nerve as described above.

Example  3.3 - Histological examination

Prior to paraffin embedding, the medial part of each penis piece was immediately fixed in 10% formalin as soon as it was recovered. The specimens were cut with 4-μm sections and stained with Masson trichrome.

For fluorescence microscopy, the penile tissue was fixed in 4% paraformaldehyde at 4 ° C for 24 hours, and frozen tissue sections (12 μm thick) were applied to PECAM-1 (platelet / endothelial cell adhesion molecule, endothelial marker; 1: 50), Phospho-eNOS (phosphorylated endothelial nitric oxide synthase, phosphorylated endothelial nitric oxide synthase, Cell Signaling Beverly, MA, USA; 1:25), smooth muscle alpha -actin (Sigma; 1: 200), nNOS (neuronal nitric oxide synthase, nerve cell-derived nitric oxide synthase, Santa Cruz Biotechnology, Santa Cruz, CA, USA; (Sigma-Aldrich Co, St. Louis, Mo., USA; 1: 100) overnight at 4 ° C. Control sections were incubated without primary antibody. After washing several times with PBS, the sections were incubated with tetramethyl rhodamine isothiocyanate- or FITC-conjugated secondary antibody for 2 hours at room temperature. Samples were stained for nuclear staining by storing them in a solution containing DAPI (4,6-diamidino-2-phenylindole, Vector Laboratories, Inc. Burlingame, Calif., USA). The signals were visualized and digital images were obtained using a confocal microscope (FV1000, Olympus, Tokyo, Japan).

The endothelial cells, smooth muscle cells and collagen content of the sponges were analyzed using a quantitative image analysis system (National Institutes of Health [NIH] Image J 1.34, http://rsb.info.nih.gov/ij/index.html) . In addition, the degree of spongy fibrosis, which is determined by the ratio of smooth muscle collagen in the cavernosal tissue, was evaluated.

Example  3.4 - Protein antigen - antibody immunity (Western blotting method)

The middle part of each penis piece is rapidly frozen in liquid nitrogen as soon as it is recovered. The rapidly frozen tissue is finely ground and the proteins are extracted using a mixture of RIPA buffer and phosphate inhibitor cocktail. Proteins are separated by size using SDS-PAGE electrophoresis and then transferred to nitrocellulose to induce an antigen-antibody immune response. (1: 300), eNOS (Transduction Laboratories Inc., Lexington, KY, USA; 1: 300), β-actin (Abcam, Cambridge, UK; 1: 6000) The cells were cultured overnight to react with the antigens in the celluloses. After washing three times with PBS buffer, the secondary antibody is incubated at room temperature for 2 hours and then washed three times with PBS buffer. The results were obtained by reacting the antigen-antibody-immune-reacted nitrocellulose with the ECL solution.

Example  3.5 - Statistical analysis

Results are expressed as mean ± standard error. Group comparisons of parametric data were made by one-way ANOVA according to the Newman-Keuls post hoc test. We obtained non-parametric data using the Kruskal-Wallis test. Statistical analysis was performed using SigmaStat 3.5 software (Systat Software Inc., Richmond, Calif.). When P was less than 5%, it was judged to be significant.

Example  4 - Blood vessel toughness  Efficacy of erectile dysfunction treatment

Example  4.1 - In diabetic erectile dysfunction mice, Clonal BMSCs  Confirmation of erectile function restoration of transplant

After 2 weeks of treatment, intracavernous pressure of the cavernous vein was measured after stimulation of the spongy nerve (5 V, 12 Hz, 1 ms) for 1 minute in a normal control or diabetic mouse, Respectively.

The ratio of ICP to total ICP for mean systolic blood pressure (MSBP) was significantly lower in the diabetic group and PBS-treated diabetic group than in the normal control group. Single IC infusion of clonal BMSCs induced recovery of erectile function compared to diabetic group and PBS-treated diabetic group, which showed recovery from 80 to 100% of normal control value. (Figs. 8B and 8C). No detectable difference in systemic blood pressure was observed between the four experimental groups.

Example  4.2 - In diabetic erectile dysfunction mice, Clonal BMSCs  Transplant Spongy through Endothelial cell  And smooth muscle cell content

Immunohistochemical staining of sponge tissue using antibodies against PECAM-1 was performed on normal control and diabetic mice two weeks after treatment. Significantly lower levels of spongy endothelial cells were observed in diabetic and PBS-treated diabetic mice than in normal control mice. IC injection of clonal BMSCs significantly restored sponge endothelial cell content (FIGS. 9A and 9B).

Two weeks after treatment, immunohistochemical staining of cavernosal tissues was performed using antibodies against smooth muscle alpha -actin in normal control and diabetic mice. Significantly lower levels of sponge smooth muscle cells were observed in diabetic and PBS-treated diabetic mice compared to normal control mice. IC injection of clonal BMSCs in diabetic mice restored sponge smooth muscle content (FIGS. 9A and 9C).

Example  4.3 - In diabetic erectile dysfunction mice, Clonal BMSCs  Through transplantation Endothelial cell origin  Induction of phosphorylation of nitric oxide synthase

Immunohistochemical staining of sponge tissues and protein antigen-antibody immunization (Western blotting method) using antibodies against phosphorylated-eNOS were performed on normal control and diabetic mice 2 weeks after treatment. Significantly lower phosphorylation of endothelium-derived nitric oxide synthase was observed in diabetic and PBS-treated diabetic mice than in normal control mice. IC injection of clonal BMSCs showed that the phosphorylation of endothelium-derived nitric oxide synthase was normal (Fig. 10).

Example  4.4 - In diabetic erectile dysfunction mice, Clonal BMSCs  Transplant Spongy through  Inhibition of fibrosis

After 2 weeks of treatment, sponge smooth muscle and collagen content were measured in normal control and diabetic mice using Masson trichrome staining. The degree of spongy fibrosis, ie, sea level collagen / smooth muscle ratio, was significantly higher in diabetic and PBS-treated diabetic mice than in normal control mice. IC injection of clonal BMSCs in diabetic mice inhibited spongy fibrosis (Figure 11). No difference in detectable spongy collagen content was observed between the four experimental groups (FIG. 11).

Example  4.5 - In diabetic erectile dysfunction mice, Clonal BMSCs  Penile nNOS through transplantation and Nerve microfiber  Increase in content

Expression of nNOS and nerve fibers in penile nerve bundles was significantly lower in diabetic and PBS-treated diabetic mice than in normal controls. IC injection of clonal BMSCs in diabetic mice significantly restored neuronal microfibrils and nNOS content (FIG. 12).

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalent forms of certain embodiments of the invention specifically set forth herein. Such equivalents are intended to be encompassed within the scope of the claims.

Claims (25)

A pharmaceutical composition for preventing or treating erectile dysfunction, comprising stem cells obtained through a subfractionation culturing method. The pharmaceutical composition according to claim 1, wherein the erectile dysfunction is arteriovenous, venous, hormonal, neurogenic. The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 1, wherein the erectile dysfunction is caused by spongiform nerve injury or diabetes. The pharmaceutical composition according to claim 1, wherein said pharmaceutical composition is administered intracavernously. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered intraperitoneally. The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 1, wherein the stem cells do not substantially affect sponge collagen content. The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 1, wherein the pharmaceutical composition increases the content of smooth surface of the penis. The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 1, wherein the pharmaceutical composition increases the content of nNOS and nerve microfibrils in the penis. The method according to claim 1,
(i) placing the cell sample in the first vessel without centrifugation so that the more dense cells are at the bottom and the less dense cells are at the supernatant;
(ii) transferring a supernatant containing cells of low density in the first vessel to the second vessel;
(iii) locating cells present in the supernatant such that the more dense cells are at the bottom and the less dense cells are at the supernatant;
(iv) repeating steps (ii) and (iii) three or more times;
(v) isolating a single colony of multi-lineage stem cells or progenitor cells from the supernatant;
(vi) transferring the cells from the colony to the growth medium and culturing the cells;
(vii) expanding the cells in 4 to 14 passages; And
(viii) obtaining a bank of multi-system stem cells or progenitor cells;
Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of erectile dysfunction. [Claim 11] The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 9, wherein the cells of step (vii) are expanded to 4 to 6 passages. [Claim 11] The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 10, wherein the cells are frozen cells after 4 to 6 passages. The pharmaceutical composition according to claim 11, wherein the freezing is frozen at -110 ° C to -150 ° C or in liquefied nitrogen. The pharmaceutical composition according to claim 11, wherein the cell further comprises thawing and expanding the cells to obtain fresh cells. [Claim 11] The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 9, wherein the cells of step (vii) are expanded in 8 to 10 passages. [Claim 16] The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 14, wherein the cells are frozen after 8 to 10 passages. 16. The pharmaceutical composition according to claim 15, wherein the freezing is frozen at -110 DEG C to -150 DEG C or in liquefied nitrogen. 16. The pharmaceutical composition according to claim 15, wherein the cell further comprises thawing and expanding the cells to obtain fresh cells. [Claim 11] The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 9, wherein the cells of step (vii) are expanded to 11 to 14 passages. [Claim 18] The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 18, wherein the cells are frozen after 11 to 14 passages. 19. The pharmaceutical composition according to claim 18, wherein the freezing is frozen at -110 DEG C to -150 DEG C or in liquefied nitrogen. 19. The pharmaceutical composition according to claim 18, wherein the cell further comprises thawing and expanding the cells to obtain fresh cells. The pharmaceutical composition for preventing or treating impotency according to claim 9, wherein the container has a flat bottom. The pharmaceutical composition for preventing or treating erectile dysfunction according to claim 9, wherein the container is coated with a cell adhesive. 24. The pharmaceutical composition according to claim 23, wherein the cell adhesive comprises a polymer of any charged amino acid. 25. The pharmaceutical composition according to claim 24, wherein the cell adhesive is collagen, polylysine, polyarginine, polyaspartate, polyglutamate or a combination thereof.

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