KR20130075027A - Composition comprising pre-differentiated amniotic fluid stem cells for treating urinary incontinence - Google Patents

Abstract

PURPOSE: A composition containing pre-differentiated amniotic fluid stem cells is provided to regenerate a muscle tissue and to be used as a therapeutic agent for treating urinary incontinence. CONSTITUTION: A cell therapeutic agent for regeneration of a muscle tissue contains amniotic fluid stem cells. The muscle tissue is urethra sphincter. The stem cells have characteristics of: positive immunity to CD44, CD73, CD90, and CD105; positive immunity to SSEA4; negative immunity to CD45; and negative immunity to MHC Class II antigens (HLA-DR).

Description

(Composition Comprising Pre-differentiated Amniotic Fluid Stem Cells for Treating Urinary Incontinence)

The present invention relates to a therapeutic agent for regenerating urethral sphincter muscle comprising pre-differentiated Amniotic Fluid Stem Cells. More specifically, the present invention relates to a therapeutic agent for urethral sphincter regenerative cell comprising a muscle, neuron, The present invention relates to a therapeutic agent for urethral sphincter regenerative cells, which comprises, as an active ingredient, an initial differentiation into endothelial cells and combining these three kinds of cells. The present invention also relates to a composition for preventing and treating urinary incontinence comprising early differentiated amniotic stem cells.

Stem cells are cells capable of self-replication and capable of differentiating into two or more cells. They are totipotent stem cells, pluripotent stem cells, and pluripotent stem cells. It can be classified as multipotent stem cells (multipotent stem cells).

Totipotent stem cells are all-in-one cells that can develop into a complete individual. Cells from the oocyte and sperm after fertilization up to 8 years of age have these properties. Can be generated as a complete entity. Pluripotent stem cells are cells that can be formed in various cells and tissues derived from ectoderm, mesoderm, and endodermal layer. The pluripotent stem cells are composed of inner cell (inner cell) located inside the blastocyst appearing 4-5 days after fertilization mass, which is called an embryonic stem cell and differentiates into various tissue cells but does not form new life forms.

Multipotent stem cells are stem cells that can only differentiate into specific cells that form tissues and organs that contain these cells. The pluripotent stem cells were first isolated in adult bone marrow (Y. Jiang et al., Nature, 418: 41, 2002) and then in many other adult tissues (CM Verfaillie, Trends Cell Biol., 12: 502, 2002). In other words, pluripotent stem cells have been identified from the skin, blood vessels, muscles, and brains in addition to bone marrow (JG Toma et al., Nat. Cell Biol., 3: 778, 2001; M. Sampaolesi et al., Science, 301 : 487, 2003; Y. Jiang et al., Exp. Hematol., 30: 896, 2002). However, stem cells in adult tissues such as bone marrow are very rare, and these cells are difficult to cultivate in an undifferentiated state, so that the pluripotent stem cells are very difficult to be preserved in vitro after isolation.

On the other hand, the amniotic stem cells of a person surrounding the fetus can be easily obtained during pregnancy or birth, can be mass-grown, and do not form a tumor when they are transplanted into an animal like embryonic stem cells. In particular, there is no ethical problem of embryonic stem cells, and thus various studies using stem cells as promising stem cells that are highly likely to be used for cell therapy have been carried out. Although stem cells, bone marrow, fat, and bone are used as stem cells of stem cells, most stem cells that can be obtained by noninvasive methods, including hemorrhage, infection, A circle is required. Therefore, when compared with embryonic stem cells, amniotic stem cells that do not form tumors upon transplantation into animals, have no ethical problems in harvesting, and have differentiation potential and ultimately can differentiate into all organs of the human body, Cell source.

On the other hand, female incontinence is caused by deflation of the urethra and bladder due to weakening of the support of the pelvic muscles due to birth and old age. At present, the number of female incontinence patients is estimated to be between 4 and 5 million, and the number of patients is increasing every year due to the rapid increase of older women. Thus, women's incontinence is becoming a serious social problem in the world. Surgery and injection therapy have been used to treat urinary incontinence to support the urethra and bladder. Currently, surgery is an invasive method that can cause complications. Injection therapy is expensive and can not be easily used for patients. In addition, the success rate is only 50 to 60% .

Therefore, the injection method of stem cells has been actively studied as a method which can easily improve urinary incontinence by regenerating the urethral sphincter without requiring general anesthesia.

As an example of a method for regenerating urethral sphincter using such stem cells, Korean Patent Laid-Open No. 10-2009-0056925 discloses a method for treating urinary incontinence using adipocyte-derived stem cells. In Korean Patent Publication No. 10-2010-0018655, The present invention relates to a method for treating a sphincter disorder comprising a cell, but does not show a satisfactory therapeutic effect.

As a result, the inventors of the present invention have found that early differentiated amniotic stem cells are effective for the treatment of urinary incontinence. In particular, amniotic stem cells are initially differentiated into muscle, nerve, and endothelium, Type cells in an appropriate ratio, the present inventors have completed the present invention by confirming that they have excellent therapeutic effect on urinary incontinence.

Korean Patent Publication No. 10-2009-0056925, 10-2010-0018655

It is an object of the present invention to provide a therapeutic agent for muscle regenerative cells comprising early differentiated amniotic stem cells.

Another object of the present invention is to provide a composition for preventing and treating urinary incontinence comprising early differentiated amniotic stem cells.

In order to solve the above problems, the present invention provides a therapeutic agent for regenerating muscle tissue comprising early differentiated amniotic fluid stem cells.

The present invention also provides a composition for preventing and treating urinary incontinence comprising early differentiated amniotic fluid stem cells.

The composition containing amniotic fluid stem cells of the present invention has excellent muscle tissue regeneration effect and is useful as a therapeutic agent for urinary incontinence.

Figure 1 shows the characteristics of human amniotic fluid stem cells for antigenic factors.
FIG. 2 shows the results of real-time PCR for early differentiation of amniotic stem cells into muscle, nerve, and endothelial cells.
Figure 3 shows the result of LPP in the urethral sphincter animal model.
Figure 4 shows the results of CP in an urethral sphincter animal model.
FIG. 5 shows the result of the examination of the capillary blood vessels in the urethra sphincter muscle and the regeneration of the Yonggun layer through H & E and IHC staining in the urethral sphincter animal model.
FIG. 6 shows the result of real-time PCR in the urethral sphincter animal model to confirm expression of genes related to muscle, nerve, and endothelial cell differentiation.
FIG. 7 shows the result of confirming the in vivo immune response of the early differentiated amniotic stem cells.
FIG. 8 shows the results of confirming in vivo safety of early differentiated amniotic stem cells.
FIG. 9 shows the results of in vivo tracing of early differentiated amniotic stem cells through MNPs @ SiO2 labeling.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention provides a therapeutic agent for muscle regenerative cells comprising early differentiated amniotic stem cells.

The muscle tissue includes the urethral sphincter.

In another aspect, the present invention provides a composition for preventing and treating urinary incontinence comprising early differentiated amniotic stem cells.

The amniotic stem cells are characterized in that they are initially differentiated from the outside of the body into muscles, nerves, and endothelial cells, and then blended in order to obtain optimal efficiency.

The mixing ratio of the muscle, nerve, and endothelial cells is not particularly limited, but is preferably 6: 1: 1 to 10: 1: 1, more preferably 8: 1: 1 as a cell number ratio.

As used herein, the term 'stem cells' refers to master cells that can be regenerated without limitation to form specialized cells of tissues and organs. Stem cells are developable pluripotent or pluripotent cells. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one derived (transit) cell, and then proliferate into mature, fully formed cells of the tissue.

The term "differentiation" as used in the present invention refers to a phenomenon in which cells and tissues specialize in structure or function during growth of cells through proliferation and proliferation, It means changing. In general, a relatively simple system is separated into two or more qualitatively different systems. For example, there is a qualitative difference between the parts of a biological system that were almost homogeneous at first, such as the distinction of heads or trunks between the eggs that were homogeneous at first in the development of an individual, or the distinction of cells such as muscle cells or nerve cells The state in which there is a difference or as a result is divided into qualitative or inferior parts or partial systems is called differentiation.

The term "cell therapeutic agent" used in the present invention is a medicament (US FDA regulation) used for the purpose of treatment, diagnosis and prevention of cells and tissues produced by isolation, cultivation and special work from human, Refers to a drug used for therapeutic, diagnostic and prophylactic purposes through a series of actions, such as alive, homologous, or xenogeneic cell proliferation selection, or otherwise altering the biological characteristics of a cell, in order to restore the cell. The cell therapy agent is classified into a somatic cell therapy agent and a stem cell treatment agent according to the degree of cell differentiation, and the present invention particularly relates to a therapeutic agent for amniotic stem cell.

The composition of the present invention may be used to treat urinary incontinence by administering it to an incontinent patient or animal. The urinary incontinence includes, but is not limited to, stress urinary incontinence, urge incontinence, first-degree incontinence, and incontinence incontinence.

The therapeutic composition comprising muscle, nerve and endothelial cells differentiated from the amniotic stem cell of the present invention or the amphibolite stem cell can be injected into a patient's body in a state where the cell is cultured alone or in an incubator. For example, Clinical methods published by Lindvall et al. (1989, Arch. Neurol. 46: 615-31) or Douglas Kondziolka (Pittsburgh, 1998) are available. The preparation may contain conventional pharmaceutically acceptable carrier besides the muscle, nerve and endothelial cells that have been differentiated from the amphibian stem cell or the amniotic stem cell in the early stage. In the case of an injectable preparation, a preservative, an anhydrous agent, a solubilizer or a stabilizer Etc., and in the case of a preparation for topical administration, a base, an excipient, a lubricant or a preservative may be included.

The pharmaceutically acceptable carriers to be included in the cell treatment agent of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, It is not. The cell therapy agent of the present invention may further comprise a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The cell treatment agent of the present invention may be manufactured in a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs Or into a multi-dose container. The formulations may then be in the form of solutions, suspensions or emulsions in oil or aqueous media, and may further comprise dispersants or stabilizers.

The composition of the present invention may be administered parenterally, intravenously, subcutaneously, intraperitoneally or topically, preferably in the form of an injection for periurethral injection. Compositions for parenteral administration (e. G., Injections) of the compositions according to the present invention can be prepared by dispersing and / or dissolving a pharmaceutically acceptable carrier, e. G., Sterile purified water, buffer at about pH 7, or physiological saline, And if necessary, may contain conventional additives such as preservatives, stabilizers and the like.

In addition, the amount of stem cells to be injected in the present invention is not limited thereto, but may be 10 ⁵ to 10 ⁶ cells / sphincter, preferably about 10 ⁶ cells / sphincter. The dosage may be prescribed in various ways, such as by the method of formulation, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient.

As used herein, the term "prophylactic " means any action that inhibits or slows the progression of urinary incontinence by administration of the composition of the present invention.

As used herein, the terms " treatment "and" improvement "refer to all actions in which the administration of the composition of the present invention improves or improves urinary incontinence.

The composition of the present invention may be administered in combination with therapies conventionally used to treat, prevent or treat urinary incontinence.

Hereinafter, the present invention will be described in detail by way of examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1: Isolation and Characterization of Human Amniotic Stem Cells

The ethics committee of Kyungpook National University College of Medicine approved the use of human amniotic fluid in connection with this study. The amniocentesis was obtained from a pregnant woman who underwent a routine amniocentesis for 15 to 19 weeks of pregnancy. The supernatant was removed by centrifugation of the obtained amniotic fluid. Cells were incubated with Chang Medium (a-MEM) containing 18% Chang B and 2% Chang C (Irvine Scientific, Irvine, Calif.), And embryonic stem cell-fetal bovine serum (Gibco-invitrogen, The cells were cultured on Petri dishes using 15% ES-FBS, 1% glutamine and 1% penicillin / streptomycin, Gibco. Unattached cells were removed at 1 week, and the attached cells occupied more than 80% , And the culture medium was changed every 3 days.

The surface antigen of cultured amniotic fluid cells (passage 3) was analyzed by Fluorescence Activated Cell Sorter (FACS, BD Biosciences, San Jose, CA) system. The characteristics of the cells include mesenchymal stem cells (SSEA4, CD44, CD73, CD90 and CD105), hematopoietic stem cells (CD45) and cell surface antigen factors (HLA-DR) (BD Biosciences, San Jose , CA) markers. Stem cell fractions were performed on a cell sorting system (MACS, Miltenyi Biotec, Germany) using a pluripotency marker, C-kit (Santa Cruz Biotechnology, Santa Cruz, Calif.).

As a result of the FACS analysis, amniotic cells in the passage 3 showed a strong positive for the mesenchymal markers (CD44, CD73, CD90 and CD105) and weak for the SSEA-4, and the hematopoietic markers (CD45) and MHC Negative response to Class II antigen (HLA-DR) (Fig. 1). In the amniotic cell group composed of various kinds of cells, a c-kit fraction was obtained to obtain one type of stem cell, and about 98.40% of positive stem cell group was obtained in the second fraction. Finally, human amniotic fluid stem cells (hAFSCs) were obtained through the above experiments.

Example  2: Early differentiation of amniotic stem cells into muscle, nerve and endothelial cells Pre - differentiation )

In order to establish optimized conditions for pre-differentiation from amniotic stem cells to three different cells, media were compared to conventional commercial medium and conditioned medium (CM). Gibco-Invitrogen's medium (Grand Island, NY) was used for myogenesis in the existing commercial medium, and the medium (Carlsbad, CA) for neurogenesis was used for neurogenesis , And Lonza's medium (Walkersville, MD) was used for endothelial genesis. CM was obtained from cultures obtained from primary cultures in human muscle tissue or blood vessels and neurons purchased from ScienCell were cultured and used. For pre-differentiation, amniotic stem cells were cultured in each medium for 7 days. Real-time PCR and immunocyte staining were performed according to a known method to confirm the differentiation of amniotic stem cells into muscle, nerve and endothelial cells.

Real-time PCR analysis revealed that the expression of early-myocyte differentiation (MYOD), neuronal differentiation (MAP2) and endothelial cell differentiation (CD31) markers was significantly increased in CM medium compared to conventional commercial medium Respectively. Gene expression was confirmed by immunocyte staining (FIG. 2). From these results, it was confirmed that amniotic stem cells were initially differentiated into muscle, nerve and endothelial cells, respectively.

Example  3: Animal experiment

3-1. Establish an animal model of urethral sphincter injury

The animals were treated according to the National Institutes of Health Animal Care Guidelines and the guidelines were approved by the Animal Ethics Committee of Kyungpook National University Medical School. All experiments used a total of 40 female ICR mice of 20 to 25 mg. To construct a mouse model with impaired urethral sphincter, all animals underwent aseptic surgery under normal anesthesia. The lower midline abdominal incision was performed, the bladder and urethra were exposed, and the horny nerves on both sides were confirmed and then transected. Another 10 mice were subjected to a sham operation as a control.

One week after surgery, mice were randomly separated and divided into five groups. Positive control group sham-operation [ctrl (+)]; Negative controls injected with pudendalneurectomy and saline [ctrl (-)]; A single group (M) injected with pre-differentiated amniotic stem cells into pudendalneurectomy and muscle cells; A complex infusion group (MN, muscle / neuron ratio 9: 1) in which early differentiated amniotic stem cells were mixed with muscle cells and neurons, a complex infusion in which early differentiated amniotic stem cells were mixed with muscle cells, nerve cells and endothelial cells (MNE, muscle / neuron / endothelium ratio 8: 1: 1) (number of mice in each group = 5). Each prepared 0.5x10 ⁶ cells was injected into the urethral sphincter using a 26G Hamilton microsyringe (Hamilton Company, Reno, NV).

3-2. Urinedynamics  inspection

The leakage point pressure (LPP) and the closing pressure (CP) of the urethra were measured using a conventional tilt / intravesical pressure clamp model at 2 and 4 weeks of cell injection. Prior to the measurement, spinal cord excision was performed at the T9 to T10 level to eliminate the reflex bladder action with increasing bladder pressure. Under normal anesthesia, the bladder was exposed through a midline incision. 50 ml of physiological saline solution was connected to the PE-90 tube, and physiological saline was gradually injected to increase the pressure in the bladder. The pressure at the start of urine leakage was defined as LPP. Thereafter, the pressure at the time point when the bladder pressure decreased and the urinary leakage stopped was defined as CP. Three LPP and CP measurements were taken for each animal.

As shown in FIG. 3, the LPP values of the ctrl (+), M, MN, MNE and ctrl (-) groups after 2 weeks of cell injection were 36.13 ± 0.38, 36.96 ± 2.60, 31.30 ± 1.38, 30.84 ± 1.79 and 20.75 + - 2.89 cm H ₂ O, respectively. The LPP values after 4 weeks of cell injection were 36.54 ± 1.17, 38.43 ± 1.85, 35.88 ± 0.26, 43.08 ± 0.07 and 26.58 ± 1.87 cm H ₂ O, respectively. From the above results, it was confirmed that the LPP value of the MNE group mixed with the early differentiated muscle, nerve and endothelial cells from amniotic stem cells was significantly higher than that of the other groups (p = 0.0005) MNE group with differentiated muscle, nerve, and endothelial cells were significantly effective in the treatment of urinary incontinence.

As shown in FIG. 4, the CP values at 2 weeks after cell injection of ctrl (+), M, MN, MNE and ctrl (-) groups were 22.68 ± 0.43, 21.84 ± 1.50, 19.89 ± 1.17, 17.20 ± 1, and it found to be 10.98 ± 2.46 cm H ₂ O. The CP values after 4 weeks of cell injection were 25.73 ± 1.08, 28.38 ± 3.09, 23.80 ± 0.86, 28.91 ± 0.58 and 16.83 ± 1.74 cm H ₂ O, respectively. From the above results, it was confirmed that the CP value of MNE group mixed with early differentiated muscle, nerve and endothelial cells from amniotic stem cells was significantly higher than that of ctrl (+) group ( p = 0.0004) The values were not significantly different from the ctrl (+) group ( p = 0.1078). It was found that MNE group, which is a mixture of early differentiated muscle, nerve and endothelial cells from amniotic stem cells, was the most effective treatment for urinary incontinence.

3-3. Histology, immunohistochemistry, and Molecular  analysis

After LPP and CP measurements mice were sacrificed to obtain urethral sphincter tissues. Tissue samples were subjected to general hematoxylin / eosin staining and immunohistochemical staining (IHC) using MyoD, ß-tubulin III and CD31 antibodies. In addition, genes involved in the differentiation of muscle cells (Pax7, Myf5, MyoD, Myogenein), neurons (vimentin, nestin, Map2, ß-tubullin III) and endothelial cells (Cd34, Cd31, Vegfr2, vWF) .

As a result, H & E staining showed normal regeneration of the lamellar layer and capillary blood vessels in the urethral sphincter. Especially, the regeneration of the urethral sphincter was accelerated in the group of muscle, nerve and endothelial cells (MNE). These results were confirmed again by IHC staining (Fig. 5). In real-time PCR, the MNE group was also expressed in muscle cells ( Pax7 , Myf5 , MyoD And Myogenin), neurons (Vimentin, Nestin, Map2 and ß-tubullin III) and endothelial cells (Cd34, Cd31, (Fig. 6, the expression of genes related to Vegfr2 and vWf) differentiation was confirmed that increase as compared to other groups) . The experimental results showed that the MNE group had the most regenerating effect on the limbic layer, and that the treatment effect of urinary incontinence was confirmed.

3-4. In vivo immune response and safety

In order to confirm the in vivo immune response of pre-differentiated cells, tissues were separated 2 weeks after the injection and IHC staining was performed using a cytotoxic T cell marker (CD8, BD Pharmigen, San Jose, CA) Respectively. Human fibroblasts were injected as a positive control group, and apparent control group was used as a negative control group. The cell body was transplanted by selecting under the renal capsule as a suitable environment for confirming the therapeutic safety of the cells in the living body. Amniotic stem cells in vitro were pre-diferentiated for 7 days and the cell bodies (1x10 ⁶ ) were transplanted into the left renal subcapsular space of ICR mice. Four weeks later, samples were collected and subjected to general H & E staining.

As a result of IHC staining, CD8 lymphocyte was rarely found in the urethral sphincter tissue of the group injected with the cells, and the level of expression was the same as the negative control group. On the other hand, CD8 lymphocytes with markedly increased expression were found in the group (ctrl (+)) injected with human fibroblasts (Fig. 7). In vivo safety analysis, histological analysis showed no formation of teratoma after 2 weeks of cell injection (Fig. 8). Therefore, it was confirmed through the above experiments that the muscle, nerve and endothelial cells differentiated from amniotic fluid stem cells of the present invention were immunologically safe in vivo and could be used as therapeutic agents.

3-5. In vivo tracing with MNPs @ SiO2 label

For in vivo tracing of injected cells, cells labeled with nanoparticles were used. 0.5 x 10 ⁶ cells per group were injected into the urethral sphincter of female nude mice (weighing 20-25 gm, purchased from the central laboratory animal), and after 14 days, a set of filters for FITC (Omega Optical, Brattleboro, VT) Pro-imaging system (Princeton Instrument, Trenton, NJ). Images were analyzed using Princeton Instrument software (winview / 32 Metavue) and spectral un-mixing algorithms were used to remove nonspecific auto-fluorescence. As a control, mice not injected with cells were used, and no fluorescence signal was observed in vivo.

FITC injected mice are easily visualized through the injection site. Fluorescence intensity in urethral sphincter tissue gradually decreased with time. Compared with single cell injection group, mixed and injected muscles, nerve and endothelial cells showed strong and concentrated images. On day 14 after injection, the fluorescence signal was lost in the single-cell injection group, whereas in the complex group in which two or more cells were mixed (FIG. 9).

Hereinafter, formulation examples of the pharmaceutical composition containing the composition of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

1. Preparation of injections

Early differentiated muscle, nerve and endothelial cells from amniotic stem cells 10 ⁵ to 10 ⁶ cells / sphincter

180 mg mannitol

Sterile distilled water for injection

Na ₂ HPO ₄ 2H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Claims (7)

Muscle tissue regenerative cell therapy comprising amniotic fluid stem cells.
The cell therapy agent according to claim 1, wherein the muscle tissue is a urethral sphincter.
The method of claim 1, wherein the amniotic fluid stem cells cell therapy, characterized in that the initial pre-differentiation of muscle, nerve or endothelial cells in vitro.
The cell therapeutic agent according to claim 3, wherein the muscle, nerve or endothelial cells are formulated in a ratio of 8: 1: 1.
The method of claim 1, wherein the amniotic fluid stem cells are characterized in that the cell therapy, characterized in that the following characteristics.
(a) exhibits positive immunological properties for CD44, CD73, CD90 and CD105;
(b) shows positive immunological properties for SSEA4;
(c) demonstrating the immunological properties of the negative for CD45; And
(d) exhibit negative immunological properties to MHC Class II antigen (HLA-DR);
Pharmaceutical composition for the prevention and treatment of urinary incontinence comprising amniotic fluid stem cells
7. The composition of claim 6, wherein the amniotic fluid stem cells are initially differentiated into muscle, nerve or endothelial cells in vitro.

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