Agent for Treating Urinary Incontinence Including Stem Cells Derived from Amniotic Fluid

Abstract

The present invention relates to a cell therapy product which is intended for regenerating a sphincter muscle and which contains stem cells derived from amniotic fluid, and more particularly, to a cell therapy product which is intended for regenerating the sphincter vesicae and which contains stem cells derived from amniotic fluid. Also, the cell therapy product of the present invention can be provided in the form of a formulation for administration through injection, said formulation being injected into a hydrogel complex to thereby improve the effects thereof. The composition including stem cells derived from amniotic fluid according to the present invention enables stem cells to be differentiated into muscles in the body of individual suffering from urinary incontinence by directly injecting the composition into the individual, thus effectively controlling urinary incontinence by recovering muscle functions. That is, the stem cells derived from amniotic fluid of the present invention are differentiated into muscles in-situ, and the differentiation into muscles can thus be achieved only with cells in order to recover muscle functions.

Claims

1-11. (canceled)

12. A method for treating a subject for urinary incontinence, the method comprising: administering directly to a sphincter deficiency area of the subject an effective amount of a hydrogel composition comprising an alginate/pluronic acid F-127 (Pluronic F-127, PF-127)/hyaluronic acid complex, into which amniotic fluid-derived stem cells are impregnated, wherein the alginate, Pluronic F-127 and hyaluronic acid are present in the hydrogel in a volume ratio of 6:6:1, respectively, such that the amniotic fluid-derived stem cells differentiate in situ into myocytes in vivo.

13. The method according to claim 12, wherein the hydrogel composition is formulated in the form of an injection formulation.

14. The method according to claim 12, wherein the administering comprises directly injecting the composition into the sphincter deficiency area of the subject.

15. The method according to claim 12, wherein the amniotic fluid-derived stem cells are administered in an amount of 10.sup.5 to 10.sup.6 cell/sphincter.

Description

DESCRIPTION OF DRAWINGS

[0025] FIGS. 1A and 1B show the characteristics of hAFSCs by FACS, in which FIG. 1A shows antigen markers expressed by stem cells derived from human amniotic fluid, and FIG. 1B shows that more than 90% Class II c-kit (+) cells are positive for c-kit.

[0026] FIG. 2A through 2E show the myogenic differentiation of hAFSCs in vitro, in which FIG. 2A shows the morphological change in different myogenic conditions, FIG. 2B shows the cell viability through CCK8 assay, FIG. 2C shows the expression of stem cell and early myogenic markers, FIG. 2D shows the results of double staining of primary antibodies and nucleic acid staining DAPI, and FIG. 2E shows the control of differentiation into myoblasts.

[0027] FIG. 3A shows the visualization of the presence, migration, and duration of injected hAFSCs, FIG. 3B shows the effects of urethral sphincter regeneration medicated by injected cells, FIG. 3C shows the results of histological analysis of urethral sphincter regeneration, FIG. 3D shows the results of immunohistochemical analysis of Nestin, MyoD, -SM actin and -actinin, FIG. 3E shows the results of real-time PCR analysis, and FIG. 3F shows the results of myogenic gene expression analysis using mouse primers.

[0028] FIGS. 4A and 4B show the results of functional immunoassay of hAFSCs, in which FIG. 4A shows the analysis results of surface phenotypes and FIG. 4B shows the result of immunohistochemical staining of urethral sphincter sections on CD8 active lymphocytes.

[0029] FIG. 5A through 5D show confocal laser scanning microscopic images of hAFSCs labeled with MNPs@SiO.sub.2 (RITC), in which FIG. 5A shows the images at various concentrations of MNPs@SiO.sub.2 (RITC), FIG. 5B shows the ratio of labeled hAFSCs in the entire cells, FIG. 5C shows the results of cytotoxicity of MNPs@SiO.sub.2 (RITC), and FIG. 5D shows optical images of nanoparticle-labeled hAFSCs.

[0030] FIG. 6 is a graph showing the increase in LPP and CP by two types of hAFSCs.

[0031] FIG. 7 is a graph showing the significantly increase in LPP and CP by hAFSCs mixed with a hydrogel containing alginate, pluronic acid and hyaluronic acid.

MODE FOR INVENTION

[0032] Hereinafter, the present invention will be described in detail with reference to the following Examples. However, these Examples are illustrative of the present invention, and the scope of the present invention is not limited to these Examples.

EXAMPLE 1

Characteristics of Human Amniotic Fluid Cells and Isolation of AFSCs

[0033] The present study and the use of human amniotic fluid was approved by the Ethics Committee of the Medical School of Kyungpook National University.

[0034] Amniotic fluids were obtained from women undergoing routine amniocentesis at a gestational age of 15 to 19 weeks after informed consent. Amniotic fluids containing cells were cultured on cover glass in Amino MAXTMII (Gibco-Invitrogen, Grand Island, N.Y.) for at least two week. Cells were harvested with trypsin/EDTA solution and cultured with Chang Medium (-MEM, 15% ES-FBS, 1% glutamine, and 1% penicillin/streptomycin, Gibco), 18% Chang B, and 2% Chang C (Irvine Scientific, Irvine, Calif.) at 37 C. under 5% CO.sub.2 in petri dishes. The cells were maintained at 70-80% confluence without any feeder layer.

[0035] The cultured amniotic fluid cells (passage 3) were analyzed by fluorescence activated cell sorter (FACS, BD Biosciences, San Jose, Calif.) using various surface antigens and cellular markers. Embryonic stem cells (c-kit, SSEA-4, Oct-3/4), mesenchymal stem cells (CD44, CD45, CD73, CD90, CD105), and immunogenic markers (HLA-ABC, -DR) were used for the characterization of the cells (all BD). Isotype immunoglobulin for each antibody was used as an internal control. Stem cell group was isolated with c-kit antibodies. C-kit (+) cells were cultured with Chang Medium in petri dishes and maintained up to 4 passages. These cells were sorted with c-kit and named human amniotic fluid stem cells (hAFSCs).

EXAMPLE 2

Differentiation of hAFSCs Into Myocytes In Vivo

[0036] Three different types of myogenic media were used to differentiate hAFSCs into myocyte-like cells: (i) myogenic medium (0.5% chick embryo extract, 10% horse serum, 1% penicillin/streptomycin, DMEM low glucose, all from Gibco-Invitrogen) treated with 3 mM 5-aza-20-deoxycytidine (5-azaC; Sigma-Aldrich, St. Louis, Mo.); (ii) myogenic medium treated with TGF- (5 ng/ml, Peprotech, Rocky Hill, N.J.); and (iii) conditioned medium (CM) (obtained from human skeletal muscle cell culture medium).

[0037] hAFSCs were seeded in culture medium at a density of 3,000 cells/cm.sup.2 and cultured with Chang medium for 24 hours. Then, the medium was replaced with myogenic medium. After 24 hour culture, the myogenic medium was replaced with myocyte culture medium. Cells were grown up to 14 days for analysis. Under 5-azaC condition, Matrigel pre-coated dishes (BD Biosciences) were used.

[0038] Cell viability at 14 days was measured using a CCK-8 assay kit (Dojindo, Japan). The genotypic and morphologic conversion of hAFSCs into myogenic lineage cells was analyzed by real-time polymerase chain reaction (PCR) and immunocytochemical (ICC) staining. Total RNAs were extracted from the cultured cells using a TRI Reagent (Invitrogen). Primers were designed using Primer Express (Applied Biosystems, Warrington, UK). The sequences of stem cell and myogenic specific markers (Pax7, Myf-5, MyoD, Desmin, Dystrophin, Myogenin, -actinin, and -SM actin) are listed in Table 1.

TABLE-US-00001 TABLE1 Gene Sequences hOct4 5-GGAGAATTTGTTCCTGCAGTGC 5-AGAACCACACTCGGACCACATC hSox2 5-TCACGCAAAAACCGCGAT 5-TATACAAGGTCCATTCCCCCG hNanog 5-GCATCCGACTGTAAAGAATCTTCA 5-CATCTCAGCAGAAGACATTTGCA hSmad2 5-GACACCAGTTTTGCCTCCAGTAT 5-TCCAGAGGCGGAAGTTCTGT hALP 5-ACGAGCTGAACAGGAACAACGT 5-CACCAGCAAGAAGAAGCCTTTG hPax7 5-GCAAATTGCTGTCCTGCTCA 5-TGAAAACTGGTCACATCTGCCT hMyf5 5-ACCGATTCACAGCCTCGAACT 5-TGTGTATTAGGCCCTCCTGGAA hMyoD 5-ACAGCGCGGTTTTTTCCAC 5-AACCTAGCCCCTCAAGGTTCAG hDesmin 5-GGAGAGGAGAGCCGGATCA 5-GGGCTGGTTTCTCGGAAGTT hDystrophin 5-CATCACATCACTCTTCCAAGTTTTG 5-CCTTGGCAACATTTCCACTTC hMyogenin 5-TGGCAGGAACAAGCCTTTTC 5-ACAGGCAGGTAGTTTTCCCCA hMEF2 5-ATTCCACCAGGCAGCAAGAA 5-GGAGTTGCTACGGAAACCACTG hMLP 5-AAGGCTCTTGACAGCACGACAG 5-TGTCCATACCCGATCCCTTTG hMHC 5-CCAGCACCTGGGCAAGTC 5-CCACAACACCAGCATAGTGAATC h-SMactin 5-CAAGTGATCACCATCGGAAATG 5-GACTCCATCCCGATGAAGGA h-actin 5-ATCGTCCACCGCAAATGCT 5-AAGCCATGCCAATCTCATCTTG mPAX7-F 5-ACCAAGCTTTCAAGTCCGCA 5-GCCTTACATTCTGGAGGATGGA mMyf-F 5-CTCTGAAGGATGGACATGACGG 5-ACTGGTCCCCAAACTCATCCTC mMyoD-F 5-TTCCGGAGTGGCAGAAAGTTAA 5-TCAAGTCTATGTCCCGGAGTGG mMyogenin-F 5-TATCCGGTTCCAAAGCCTCTG 5-GCGGCAGCTTTACAAACAACA mMEF2 5-AACCCCAATCTTCTGCCACTG 5-ATCAGACCGCCTGTGTTACCTG mMLP 5-GCTGAACAAGTTACTGAGCGGC 5-ATTTTGCACCTCCACCCCA mMHC 5-CCCCGCCCCACATCTT 5-GATTGACTGATTCTCCCTGTCTGTT mGAPDH 5-TGTGTCCGTCGTGGATCTGA 5-CCTGCTTCACCACCTTCTTGA

[0039] Analysis was performed using the ABI Prism Sequence Detection System 7500 (PE Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif.). Temperature cycling conditions were based on the inset conditions, and the relative quantification was performed by the CT method. The results were normalized to beta-actin levels.

[0040] For immunocytochemistry (ICC), the cultured cells were fixed with 4% paraformaldehyde for 5 minutes. The cells were washed with PBS three times and blocked in 5% BSA for 1 hour to prevent non-specific antibody binding. Then, the cells were cultured with primary antibodies at 4 C. overnight, washed with PBS three times, and incubated with secondary antibodies at room temperature for 1 hour. The antibodies used were Nestin, MyoD, -SM actin, -actinin (all from Santa Cruz Biotechnology, Santa Cruz, Calif.) and Desmin (BD Biosciences). The secondary antibodies bound to Alexa Fluro 594 immunofluorescence were applied for 30 minutes and washed with PBS. Samples were mounted in ProLong Gold antifade reagent (Invitrogen) with 4,6-diamino-2-phenylindole (DAPI) for staining. C2C12 cell line was used as a positive control and human fibroblasts were used as a negative control.

EXAMPLE 3

Incapacitation of Urethral Sphincter and Injection of hAFSCs

[0041] Mice were treated according to National Institutes of Health Animal Care Guidelines, which were approved by the Animal Ethics Committee of the Medical School of Kyungpook National University. All experiments were performed using 4-week-old female ICR mice (2025 mg). Before the surgical injury to the urethral sphincter, the abdominal leak point pressure (LPP) and closing pressure (CP) were measured. Immediately, anesthesia was induced by intramuscular injection of Zoletil (30 mg/kg, virbac animal health, France) and Rumpun (10 mg/kg, Bayer, Korea). A lower midline abdominal incision was performed and the pudendal nerves on both sides were found. The bilateral pudendal nerves were transected with surgical scissors under a microscope.

[0042] hAFSCs (110.sup.6) were injected using a 26 G Hamilton microsyringe (Hamilton Company, Reno, Nev.) with microscopic guidance. Three experimental groups were established: a control group (Ctrl) underwent sham-operation and cell injection without neurectomy; a group Cell () underwent pudendal neurectomy and injection of saline; and a group Cell (+) underwent pudendal neurectomy and injection of hAFSCs.

EXAMPLE 4

Preparation of Hydrogel for Injection of hAFSCs

[0043] An alginate solution (3%) was prepared by dissolving 300 mg of alginic acid sodium salt in 10 ml of PBS (pH 7.4) and completely mixed using a homogenizer. To prepare a Pluronic F-127 (PF-127) solution (25%), 5 g of polymer was dispersed and stirred in 20 ml of PBS. Partially dissolved PF-127 solution was stored in a refrigerator at 0 to 4 C. or using an ice bath until the entire polymer was completely dissolved. 20 g of hyaluronic acid was dissolved in 1000 l of distilled water. The mixing ratio of alginate/PF-127/hyaluronic acid was 6:6:1, and the mixture was homogeneously mixed for 5 minutes and left in a refrigerator for 6 hours. A sodium hyaluronate solution was prepared by dissolving 100 mg of sodium hyaluronate in 10 ml of PBS by the same process. Finally, Ca.sup.2+ (CaCl.sub.2, 0.2%) was prepared by dissolving 100 mg of CaCl.sub.2 in 50 ml of PBS. To prepare a cell-containing polymer solution, amniotic fluid stem cells at 110.sup.6 were slowly mixed and added to alginate/PF-127/hyaluronic acid.

EXAMPLE 5

Urodynamic Test

[0044] Leak point pressure (LPP) and closing pressure (CP) were measured at one, two and four weeks after injection using the vertical tilt/intravesical pressure clamp model. The spinal cords of anesthetized animals were transected at the T9 level. A catheter with a fire-flared tip (PE-90) was inserted into the bladder dome, and the abdominal wall was sutured. The mice were then placed on a tilt table in the vertical position, and saline was instilled into the bladder. The intravesical pressure was increased in 13 cm H.sub.2O steps from 0 cm H.sub.2O upward until visual identification of the leak point height. The averages of three consecutive LPP and CP measurements were taken.

EXAMPLE 6

Histological, Immunohistochemical, Molecular, and Immune Response Analysis

[0045] After the urodynamic test, the urethras were harvested for each time. Injected human cells were identified using anti-human nuclear antibody (HuNu, Chemicon) and the stem/myogenic lineage cells present in the urethral sphincter were analyzed. To determine an appropriate cell number for injection, 10.sup.4, 10.sup.5, and 10.sup.6 cells/sphincter were used. Optimal cell number was determined through the analysis of stem cells and myogenic antibodies (Nestin, Myod, and -SMA). Through the histological analysis, in vivo tumor formation was also observed. The differentiation of injected hAFSCs into myocytes was determined through real-time PCR using human primers, and the host reaction required for cell therapy was measured using mouse myogenic primers. Protein expression was determined by IHC using Nestin, Myod, -SM, and -actinin antibodies after two weeks and four weeks. To evaluate the immunogenicity of injected hAFSCs, the expression of HLA-DR on the cell surface was determined using FACS and T lymphocyte activation marker (CD8) IHC at one week after injection.

EXAMPLE 7

MNPs@SiO.SUB.2 .Labeling of hAFSCs and In Vivo Tracking Through Optical Images

[0046] Magnetic nanoparticles were used to track the injected hAFSCs. Cobalt ferrite silica core-shell nanoparticles containing RITC [MNPs@SiO.sub.2 (RITC)] were provided by Dr. Jaesung Bae (Kyungpook National University, Daegu, Korea). For labeling, hAFSCs (10.sup.4) were cultured in 24 wells to reach 70% confluence, and MNPs@SiO.sub.2 (RITC) nanoparticles were added for 24 hours. To establish effective uptake of nanoparticles, various concentrations were applied such as 0.01, 0.05, 0.1 or 0.2 mg/mL. To evaluate time-dependent labeling efficiency, ICC staining was performed every 6 hours, and in vivo localization of MNPs@SiO.sub.2 (RITC) was measured. To analyze the localization of nanoparticles labeled in hAFSCs in vitro, hAFSCs at 510.sup.4 cells were seeded in 35-mm tissue culture dishes containing growth media. When the cells reached about 70% confluence, MNPs@SiO.sub.2 (RITC) (0.1 mg/mL) was added, cultured for 3 hours, and suspended in FACS buffer (n=3). Labeled cells were passaged seven times to measure the maintenance ratio in the next passages. For optical imaging, cells were labeled with MNPs@SiO.sub.2 (RITC) (0.2 mg/mL) at 37 C. for 3 hours. After anesthesia, 110.sup.6 nanoparticle-labeled cells were injected into urethral sphincter mice (n=3). After injection, optical images were obtained using Pro imaging system (Princeton Instrument, Trenton, N.J.), and filters (Omega Optical, Brattleboro, Vt.) were set for RITC. Images were analyzed using Princeton Instrument software (winview/32 Metavue), and spectral unmixing algorithms were used to eliminate non-specific autofluorescence. The effect of the concentration (0.010.2 mg/mL) of nanoparticles on the cell viability was measured using MTS assay.

[0047] [Results]

[0048] 1. Characterization of Human Amniotic Fluid Cells and Isolation of c-kit (+) cells

[0049] Cells were isolated from four different amniotic fluid samples and analyzed by FACS system (FIG. 1A). Human amniotic fluid cells expressed embryonic stem cell markers such as Oct-4, c-kit, and SSEA-4, from which it is interpreted that these cells have pluripotential capability. These cells were positive for mesenchymal stem cell markers, neural stem cell markers, and/or endothelial progenitor cell markers (CD44, CD73, CD90 and CD105), but negative for the hematopoietic lineage marker (CD45). In the case of immune response-related markers, cells were negative for Class II major histocompatibility antigen (HLA-DR). Class I c-kit (+) cell population was 0.6-5.0% of the entire cell group. More than 90% of Class II c-kit (+) cells were positive for c-kit.

[0050] 2. Myogenic Characteristics of hAFSCs In Vitro

[0051] It was found that when hAFSCs at passage 5 were cultured in regular myogenic media) (5-azaC condition) for 7 days, cells expressed myogenic specific markers. The following two additional conditions were evaluate to determine the differentiation of hAFSCs into myocytes: TGF- and conditioned medium. The cultured cells were morphologically similar such as elongated morphology (FIGS. 2A-a, b, and c). In the cell viability assay through MTS assay, the hAFSCs cultured with 5-azaC and TGF- showed significant cytotoxicity compared to CM (FIG. 2B). These results indicate that the culture of hAFSCs in CM showed mild myogenic conversion. In the real-time PCR analysis (FIG. 2C), stem cell and myogenic lineage markers in three different media showed various gene expression levels. At 3 days after culture, the expression of stem cell and early myogenic markers increased, and at 7 days, the expression of the mid to late myogenic markers was dominant. These results indicate that hAFSCs can differentiate into myogenic lineage cells in proposed media. Although the group treated with 5-azaC and TGF- showed relatively high myogenic gene expression, it is considered that the CM medium is an appropriate myogenic condition for hAFSCs in terms of cell viability. Immunohistochemical (IHC) staining was performed to determine the results of gene expression (FIG. 2D).

[0052] More than 90% of the cultured hAFSCs were stained positively for myogenic markers. Nestin was expressed with stem cell markers for the entire culture period. Cells cultured for 3 days expressed myogenic markers at the same time. At 7 days, the early markers such as MyoD and Desmin became weak, while the late markers such as -SM actin and -actinin were more expressed than before. Characteristically, the cells expressed significantly -actinin-labeled thick filaments, and cells with spindle shapes were found in the group treated with CM. C2C12 cell line was used as a positive control and human fibroblasts were used as a negative control for differentiation into myoblasts (FIG. 2E).

[0053] 3. Identification of Human Cells and Optimal Cell Number In Vivo

[0054] Human nuclear-specific antibody (HuNu) was used to determine the presence, migration, and duration of injected human cells. Human nuclei stained with anti-HuNu were matched with DAPI in vitro (FIG. 3A-a). The presence of human nuclei injected into the urethral sphincter was not found in vivo (FIG. 3A-b). Positive staining was locally observed at the injection site at 3 days, and the intensity decreased gradually and lasted up to 14 days. No HuNu-positive cells were found in cell walls of PBS-injected urethral sphincter.

[0055] Among three treated groups with different cell numbers, the group with 10.sup.6 injected cells formed muscle bundles and showed effective urethral sphincter regeneration (FIG. 3B). The presence of a large amount of Nestin, MyoD and -SM indicates the enhanced regeneration in the injured urethral sphincter according to the cell number. The group in which no cells were seeded and the group in which a small amount of cells were seeded showed insufficient regeneration without muscle tissues, limited smooth muscle bundle formation, and some irregular flaps on the outside of transitional epithelium. Due to technical limitations, the injection of 10.sup.7 cells was impossible. Thus, the optimal cell number in the mouse models was determined as 10.sup.6 cells.

[0056] 4. Histological, Immunohistochemical, Molecular In Vivo Analysis

[0057] An additional study was performed using a reasonable cell number. Mouse urethral sphincter included smooth muscles and skeletal muscles (FIG. 3C-a). Urethral sphincter area injured by neurotomy was determined as an atrophic tissue, like contracted muscles, and had no smooth muscle and straight muscle laminar structure at 2 weeks and 4 weeks (FIGS. 3C-b and c). The cell-injected group showed the regeneration of circular muscle mass and thick packed layers (FIGS. 3C-d and e). The cell-transplanted organ maintained the pattern and effect of normal urethral sphincter shape. The increased urethral sphincter in all experimental groups was considered as evidence of complete muscle regeneration. These results are histological evidence of the cellular therapeutic effects on hAFSCs injected into urethral sphincter mouse models. No tumors were found in the cell (+) group, which indicates that the transplanted hAFSCs can adjust the cell growth in in vivo conditions and maintain normal phenotype.

[0058] Stem cell and myogenic markers were determined using IHC. At 2 weeks, Nestin, MyoD, and -SM increased in the cell-injected group, and -actinin-positive cells formed the layered muscle structures showing the normal urethral sphincter structure (FIG. 3D). At 4 weeks, in the cell (+) group, the expression of the early markers (Nestin and MyoD) on the smooth and straight muscle layer decreased, and the late marker (-SM and -actinin)-positive cells were continuously maintained. On the contrary, in the cell () group, these proteins were weakly expressed. In the real-time PCR analysis, the injected human stem cells continuously expressed myogenic lineage-related genes (FIG. 3E). Gene expression using human primers relatively increased in the early stage, but decreased gradually over time. Although the injected cells had stemness properties, these cells differentiated into myogenic lineage cells in in vivo conditions. The expression of myogenic genes was analyzed using mouse primers, and the cell-treated group increased the expression of mouse genes compared to the cell () group (FIG. 3F). At 4 weeks, most of specific genes were highly expressed from the early to late markers. These results are considered that hAFSCs affect the host cell differentiation through unclear mechanism.

[0059] 5. Immune Tolerance

[0060] To analyze the functional immune, surface phenotypes of hAFSCs were analyzed using phycoerythrin-conjugated antibody against HLA-DR. hAFSCs weakly expressed HLA-DR (0.26%, 2.29%), and there was no significant difference in surface phenotype compared to samples, while there was a small change (FIG. 4A). The average ratio of positive cells was lower than that of the isotype Ab control. hAFSCs were injected into mouse urethral sphincters and active CD8 lymphocytes were stained at 1 week after injection (FIG. 4B). In the cell-injected group, CD8 lymphocytes (red) showed significantly weak reaction at the injection site, which indicates that hAFSCs did not induce immune reaction. On the contrary, the urethral sphincter which was denervated and into which human fibroblasts were injected showed significant CD8 lymphocyte reaction.

[0061] 6. Optical Imaging for In Vivo Tracking of Injected Cells

[0062] The optimum concentration of MNPs@SiO.sub.2 (RITC) for hAFSCs, which provided sufficient images in vitro, was determined at 0.2 mg/ml using a fluorescence microscope (FIG. 5A). The signal intensity increased over time, and reached the highest value at 24 hours, and then there was no significant change in intensity up to 72 hours. With respect to the labeling efficiency at 24 hours, 94.31% of the cells in the entire group showed the RITC signal (FIG. 5B). Nanoparticles were localized in cytoplasm and clearly detected in a merged image. MTS cell proliferation assay showed that the viability of hAFSCs treated with 0.01, 0.05, 0.1 and 0.2 mg/mL MNPs@SiO.sub.2 (RITC) for 24, 48 and 72 hours was similar over the entire period, which indicates that MNPs@SiO.sub.2 (RITC) did not induce cytotoxicity in hAFSCs (FIG. 5C). Referring to the effects of nanoparticles on the cell cycle progression in hAFSCs, no nanoparticle-induced cell cycle arrest was found. MNPs@SiO.sub.2 (RITC)-labeled cells were injected into the urethral sphincter (110.sup.6 cells). Optical images show that signals were expressed in the right side of the injection site of the labeled cells, while the control group and the non-labeled cells were not expressed. Over time, the signal intensity decreased slowly until 10 days (FIG. 5D).

[0063] 7. Urodynamic Test

[0064] At one week, in the Ctrl, Cell () and Cell (+) groups, the LPP was 30.252.56 cmH.sub.2O, 16.552.10 cmH.sub.2O, and 17.900.49 cmH.sub.2O, and the CP was 19.452.67, 9.130.87, and 9.881.34 cmH.sub.2O. At two weeks, the LPP was 28.670.72, 11.591.18, and 18.062.78 cmH.sub.2O, and the CP was 16.992.20, 6.851.09, and 12.421.71. At four weeks, the LPP was 27.593.64, 15.242.10, and 20.243.25 cmH.sub.2O, and the CP was 15.381.64, 8.351.10, and 14.43.40. After one week of treatment, there was no difference in LPP and CP in the Cell () and Cell (+) groups. On the contrary, at two weeks and four weeks, the LPP and CP in the Cell (+) group were significantly higher than those of the Cell () group (P=0.05) (FIG. 6).

[0065] Moreover, the cells mixed with a hydrogel complex comprising alginate, pluronic acid, and hyaluronic acid showed significant restoration of LPP and CP compared to the cell group (FIG. 7)

[0066] The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.