A COMPOSITION COMPRISING MESENCHYMAL STEM CELLS FOR INHIBITING ADIPOGENESIS

Abstract

Provided is a composition for inhibiting, preventing, alleviating, or treating adipogenesis. Mesenchymal stem cells according to one aspect or a composition comprising the same inhibit fat accumulation or adipose tissue increase and thus may be advantageously utilized in the prevention, treatment, or alleviation of diseases associated with abnormal fat metabolism.

Claims

1. A mesenchymal stem cell secreting phosphatase of regenerating liver-1 (PRL-1) or overexpressing PRL-1 compared to a parent cell.

2. The mesenchymal stem cell of claim 1, wherein the PRL-1 is transferred by a method and expressed, the method being selected from microinjection, electroporation, a DEAE-dextran treatment transfection method, lipofection, a nanoparticle-mediated transfection method, a protein transduction domain-mediated transfection method, a calcium phosphate (CaPO.sub.4) transfection method, a virus-mediated gene transfer method, and a PEG-mediated transfection method.

3. The mesenchymal stem cell of claim 1, wherein the mesenchymal stem cell is an umbilical cord-derived mesenchymal stem cell, an umbilical cord blood-derived mesenchymal stem cell, a bone marrow-derived mesenchymal stem cell, a placenta-derived mesenchymal stem cell, or an adipose-derived mesenchymal stem cell.

4. A pharmaceutical composition for preventing or treating a disease associated with abnormal adipogenesis or abnormal fat metabolic dysfunction, the pharmaceutical composition comprising a mesenchymal stem cell secreting phosphatase of regenerating liver-1 (PRL-1) or overexpressing PRL-1 compared to a parent cell.

5. The pharmaceutical composition of claim 4, wherein the disease associated with abnormal adipogenesis or abnormal fat metabolic dysfunction is selected from obesity, diabetes, dyslipidemia, metabolic diseases, hypertension, thyroid-associated ophthalmopathy, and degenerative diseases associated with abnormal adipogenesis or abnormal fat metabolic dysfunction.

6. The pharmaceutical composition of claim 4, wherein the mesenchymal stem cell inhibits fat accumulation or adipose tissue increase.

7. The pharmaceutical composition of claim 4, wherein the mesenchymal stem cell is an umbilical cord-derived mesenchymal stem cell, an umbilical cord blood-derived mesenchymal stem cell, a bone marrow-derived mesenchymal stem cell, a placenta-derived mesenchymal stem cell, or an adipose-derived mesenchymal stem cell.

8. A health functional food for alleviating or preventing a disease associated with abnormal adipogenesis or abnormal fat metabolic dysfunction, the health functional food comprising a mesenchymal stem cell secreting phosphatase of regenerating liver-1 (PRL-1) or overexpressing PRL-1 compared to a parent cell.

9. A method of inhibiting fat accumulation, the method comprising: administering a mesenchymal stem cell to a test animal in vivo or contacting the mesenchymal stem cell with cells in vitro, the mesenchymal stem cell secreting phosphatase of regenerating liver-1 (PRL-1) or overexpressing PRL-1 compared to a parent cell.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 is a schematic diagram illustrating a PRL-1 gene transferred into mesenchymal stem cells.

[0039] FIG. 2 is a microscopic photograph confirming, through GFP, whether the PRL-1 gene has been transferred into placenta-derived mesenchymal stem cells.

[0040] FIG. 3 is a graph showing results of confirming the number of PRL-1 gene-transferred placenta-derived mesenchymal stem cells (GFP+ cells).

[0041] FIG. 4 illustrates results of confirming, through RT-PCR, an mRNA expression level of PRL-1 expressed by mesenchymal stem cells according to one aspect.

[0042] FIG. 5 illustrates results of confirming, through RT-PCR, expression levels of cell surface antigens (Oct4, Nanog, Sox2, HLA-G, TERT) of mesenchymal stem cells according to one aspect.

[0043] FIG. 6 illustrates results of confirming, through ELISA, expression levels of cell surface antigens (Oct4, HLA-G) of mesenchymal stem cells according to one aspect.

[0044] FIG. 7 is a graph confirming a doubling time of mesenchymal stem cells according to one aspect.

[0045] FIGS. 8 and 9 illustrate results of analyzing, through FACS, the characteristics of cell surface antigens (CD34, CD13, CD90, CD105, HLA-DR, HLA-ABC, and HLA-G) of mesenchymal stem cells according to one aspect.

[0046] FIG. 10 illustrates results of confirming the expression of cell surface antigen markers and osteocyte and adipocyte markers in placenta-derived MSCs with enhanced PRL-1 expression when induced to differentiate to osteocytes and adipocytes.

[0047] FIG. 11 illustrates results of confirming, through co-culture, the effect of mesenchymal stem cells according to one aspect on adipogenesis of orbital fibroblasts; OF: Normal subject; TAO: Subject with thyroid-associated ophthalmopathy; CP(−): Not co-cultured; CP Naive (+): Co-cultured with placenta-derived mesenchymal stem cells; and CP-PRL-1 (+): Co-cultured with placenta-derived mesenchymal stem cells overexpressing CP-PRL-1.

[0048] FIG. 12 illustrates results of confirming, through co-culture, the ability of mesenchymal stem cells according to one aspect to regulate adipogenesis-associated genes of orbital fibroblasts; OF: Normal subject; and TAO: Subject with thyroid-associated ophthalmopathy.

[0049] FIG. 13 illustrates results of confirming, through co-culture, the influence of mesenchymal stem cells according to one aspect on adipogenesis-associated factors and inflammation-associated factors of orbital fibroblasts; TAO: Subject with thyroid-associated ophthalmopathy.

MODE OF DISCLOSURE

[0050] Hereinafter, the present disclosure will be described in greater detail by way of examples.

[0051] However, the following examples are only for illustrating one or more specific examples, and the scope of the present disclosure is not limited to the following examples.

REFERENCE EXAMPLES

Reference Example 1

Culturing and Treatment of Orbital Fibroblasts

[0052] Orbital fibroblasts were collected from human subjects (4 normal subjects, 4 patients) and cultured in DMEMF12 (Gibco) (10% FBS and 1% penicillin-streptomycin). 2 days after the fibroblasts were distributed and grown in media, 5 μg/ml of insulin, 1 mM dexamethasone, and 0.5 mM IBMX were added to DMEM (10% FBS) to initiate differentiation to adipocytes (day 0).

[0053] After 72 hours (day 3), the media were switched with DMEM media supplemented with 10% FBS and 5 μg/ml insulin, and DMEM media supplemented with 10% FBS were fed every two days thereafter.

Reference Example 2

[0054] Real-Time PCR On day 8 of culturing, placenta-derived mesenchymal stem cells with overexpressed PRL-1 (2×10.sup.5) were co-cultured with orbital fibroblasts for 48 hours.

[0055] Cell lysates were homogenized using the Trizol Reagent (Invitrogen, Carlsbad, Calif., USA) to extract RNA. From each sample, 1 μg of total RNA was reverse-transcribed to synthesize cDNA. The cDNA synthesis conditions were as follows: RNA denaturation (65° C., 1 minute), unwinding (25° C., 5 minutes), amplification (42° C., 60 minutes), and enzyme deactivation (85° C., 1 minute).

[0056] The mRNA expression of each gene was amplified under the following PCR conditions and normalized: initial denaturation (95° C., 2 minutes), amplification (95° C., 10 seconds; 55° C., 20 seconds; and 72° C., 20 seconds), 40 cycles. PPARγ, ADIPONECTIN, C/EBPα, primer sets used are shown in Table 1.

TABLE-US-00001 TABLE 1 Markers Genes Primer sequence (5′-3′) Ligation temperature (° C.) SEQ ID NO. Stemness 04-Oct F: AGT 52 1 R: GAG GTG AGG CAA AAG CCT GGA TGA GA GGG CTC CCA TA  2 Nanog F: TTC TTG ACT GGG ACC 52  3 TTG TC R: GCT TGC CTT GCT TTG  4 AAG CA Sox-2 F: AGA ACC CCA AGA TGC 52  5 ACA AC R: GGG CAG CGT GTA CTT  6 ATC CT HLA-G F: GCG GCT ACT ACA ACC 58  7 AGA GC R: GCA CAT GGC ACG TGT  8 ATC TC TERT F: GAG CTG ACG TGG AAG 55  9 ATG AG R: CTT CAA GTG CTG TCT 10 GAT TCC AAT G Osteocytic OC F: CAC CCC 58 11 TCC TCG R: CCC TAT TCC TGG C TGC TTG GAC ACA AAG 12 Col 1 F: AGA CAT CCC ACC AAT 60 13 CAC CT R: CGT CAT CGC ACA ACA 14 CCT Adipocytic Adipsin F: CAC 55 15 GTA CCA R: TGA TGG TCG GGC AA AGA TCC CCA CGT AAC CA 16 PPARg F: GAC AGA CCT CAG GCA 55 17 GAT TG R: GTC AGC GAC TGG GAC 18 TTT TC Adiponectin F: GAC TGC CAC TAA TTC 55 19 AGA GC R: CTC ATG GGG ATA ACA 20 CTC AG LPL F: ACA GGT GCA ATT CCA 55 21 AGG AG R: CTT TCA GCC ACT GTG 22 CCA TA Leptin F: ATC TAT GTG CAC CTG 55 23 AGG GTA G R: TCC TTT TCA CAA AGC 24 CAC ACT AT FABP4 F: ACA TGA AAG AAG TGG 55 25 GAG TTG GC R: AAG TAC TCT CTG ACC 26 GGA TGA CG C/EBPa F: TGT ATA CCC CTG GTG 55 27 GGA GA R: TCA TAA CTC CGG TCC 28 CTC TG — PRL-1 F: TAC 60 29 TGC TCC R: ACC AAG AGG AAG CC TTT ACC CCA TCC AGG TC 30 Internal human GAPDH F: GCA 55 control 31 CCG TCA R: group AGG CTG GTG AGA AC GTG AAG ACG CCA GTG GA 32 rat F: TCC CTC AAG ATT GTC 55 33 GAPDH AGC AA R: AGA TCC ACA ACG GAT 34 ACA TT The Data was compared to a normal Reference Using mRNA group and expressed as folds Example 3 RIPA expressions (mean ± SEM) of an adipose Western buffer, of the differentiation-associated Blot lysates respective factor. were genes prepared. were Equivalent amounts of total The normalized proteins were separated by membrane to 18s SDS-PAGE and transferred to a was rRNA. membrane. diluted to 1:1,000 in anti- HAS1 and HAS2 (Santa Cruz Biotechnology, SA, USA) and subjected to immuno- blotting, and the same membrane was cultured with GAPDH (Santa Cruz).

[0057] After rinsing, the membrane was diluted to 1:1,000 and cultured at room temperature for 3 hours with horseradish peroxidase-conjugated anti-goat IgG secondary antibody. Immunoreactive bands were imaged with an enhanced chemiluminescence solution (Animal Genetics, Suwon, Korea) and detected with ChemiDoc™ XRS+ System Imager (Bio-Rad Laboratories, Hercules, Calif., USA).

[0058] Protein expression amounts were normalized to GAPDH. Data was compared to a normal group and expressed as folds of HAS2 (mean±SEM).

Reference Example 4

[0059] FACS Analysis Human fibroblasts (3×10.sup.5) were dissociated in a cell dissociation buffer (Life Technologies) and rinsed with PBS (2%(v/v) FBS). The resulting cells were cultured with an isotype control IgG or an antigen-specific antibody (BD Biosciences, CA, USA) for 20 minutes, and used to identify cells. FACS sorting was performed using the FACS vantage Flow Cytometer (BD Biosciences, CA, USA). Example 1. Preparation of Functionally-Enhanced Mesenchymal Stem Cells 1.1. Isolation of Placenta-Derived Mesenchymal Stem Cells After obtaining an informed consent from a healthy mother who had given a normal birth, tissues were isolated from placental tissues collected from the placenta at the time of the normal birth.

[0060] The isolated tissues (chorioamniotic membranes) were placed in a 50 ml tube and had the remnant blood removed therefrom with the addition of DPBS, and subsequently, suspended matter was collected to one side by scratching the inner lining of the chorioamniotic membranes with a slide glass sterilized in 20 ml of enzyme solution I (1 mg/ml collagenase type I, 2 mg/ml Trypsin, 20 mg/ml DNase I, 1.2 U/ml Dispase, x1 PS in HBSS). After adding 10 ml of enzyme solution I and thoroughly mixing the resulting solution, enzymatic reactions were twice repeated each for 15 minutes to separate stem cells from the tissues.

[0061] The isolated cell suspension was separated by centrifugation, and the isolated cells were cultured using DMEM/F12 supplemented with 10% fetal bovine serum, 1 ug/ml heparin, and 25 ng/ml fibroblast growth factor-4 (FGF-4). Subsequently, the culture media were changed every 4 to 5 days, and subculturing was performed by treating the first subculture with TrypLE, manufactured by Invitrogen, in a short time (3 minutes) in an incubator at 37° C. 1.2. Preparation of Placenta-Derived Mesenchymal Stem Cells with Enhanced Expression of PRL-1 Gene Electroporation was used to enhance the expression of PRL-1 gene in the placenta-derived mesenchymal stem cells isolated in section 1.1. above.

[0062] In detail, a gene having the structure shown in FIG. 1 was transferred into the mesenchymal stem cells by means of electroporation. Chorionic plate-derived mesenchymal stem cells (CP-MSCs) were cultured in MEM-alpha media supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 25 ng/ml FGF-4, and 1 ug/ml heparin, in an incubator at 37° C. under CO.sub.2.

[0063] Cells were rinsed with phosphatase buffered saline (PBS) and then treated with trypsin at 37° C. for 2 minutes to detach the cells, and the cells detached using the PBS were collected and subjected to centrifugation at 1,200 rpm for 5 minutes. The pellets thus obtained were suspended with the addition of 1 ml of culture broth, and cell counting was performed using a hemocytometer.

[0064] After subjecting the collected cells to centrifugation at 200 g for 10 minutes and adding Nucleofector to suspend the cells (5×10.sup.5/100 ul), 2 ug of DNA plasmids containing PRL-1 was added. The cells were transferred into a cuvette and placed into a Nucleofection machine, and a ‘U-23’ program was run. Immediately upon termination of the program, a culture dish containing new media was stabilized in the 37° C., CO.sub.2 incubator. After 4 hours, the media in the culture dish were removed using an aspirator, and the attached cells were cultured in media containing 1.5 mg/ml neomycin (10% FBS) and in MEM-alpha media supplemented with 1% penicillin-streptomycin, 25 ng/ml FGF-4, and 1 ug/ml heparin, to yield PRL-1 gene-transferred CP-MSCs.

[0065] FIG. 2 is a microscopic photograph confirming, using GFP, whether the PRL-1 gene had been transferred into placenta-derived mesenchymal stem cells. FIG. 3 is a graph showing a result of confirming the number of PRL-1 gene-transferred placenta-derived mesenchymal stem cells (GFP+ cells).

[0066] As shown in FIG. 2 and FIG. 3, it was found that there was a significant increase in expression of the PRL-1 gene artificially transferred into the enhanced placenta-derived mesenchymal stem cells prepared in section 1.1., compared to in naive placenta-derived mesenchymal stem cells serving as a control group with no transferred PRL-1 gene.

[0067] Therefore, it was confirmed that the placenta-derived mesenchymal stem cells with enhanced expression of PRL-1 gene were successfully produced by the method described in section 1.1.

[0068] 1.3. Analysis of Characteristics of Placenta-Derived Mesenchymal Stem Cells with Enhanced Expression of PRL-1 Gene To analyze the characteristics of mesenchymal stem cells confirmed in sections 1.1. and 1.2. above, the characteristics of cytokine secretions and the characteristics of cell surface antigens were analyzed. In detail, the expression level of PRL-1 in the placental-derived mesenchymal stem cells was confirmed through RT-PCR analysis, and results thereof are shown in FIG. 4. FIG. 4 shows the mRNA expression level of PRL-1 expressed in cells. As shown in FIG. 4, the results of RT-PCR analysis showed that the placenta-derived mesenchymal stem cells (p1, p6) prepared in section 1.1. had a significantly higher mRNA expression level of PRL-1, compared to the naive cells. In addition, the expression levels of cell surface antigens (Oct4, Nanog, Sox2, HLA-G, and TERT) in the placenta-derived mesenchymal stem cells were confirmed through RT-PCR analysis, and results thereof are shown in FIG. 5. In addition, the expression levels of cell surface antigens (Oct4, HLA-G) in the placenta-derived mesenchymal stem cells were confirmed through ELISA analysis, and results thereof are shown in FIG. 6.

[0069] As shown in FIGS. 5 and 6, RT-PCR and ELISA analyses demonstrated that the placenta-derived mesenchymal stem cells (PRL1+) prepared in section 1.1. expressed the stem cell markers, Oct4, Nanog, Sox2, HLA-G, and TERT.

[0070] In addition, a doubling time at which the number of cells were doubled was confirmed by counting increases in the number of cells over cell culture processes, and results thereof are shown in FIG. 7.

[0071] As shown in FIG. 7, it was found that there was no large difference in terms of the doubling time over sub-culture processes between the PRL-1 overexpressed cells and the control group that was not induced for PRL-1 expression.

[0072] Further, the characteristics of cell surface antigens (CD34, CD13, CD90, CD105, HLA-DR, HLA-ABC, and HLA-G) of the placenta-derived mesenchymal stem cells were analyzed through FACS analyses, and results thereof are shown in FIGS. 8 and 9.

[0073] As shown in FIGS. 8 and 9, the mesenchymal stem cell markers, CD34, CD105, HLA-ABC, and HLA-G were found to be positive. Accordingly, it was confirmed that as the expression of PRL-1 was enhanced, the placenta-derived mesenchymal stem cells, which express and include PRL-1, had maintained the characteristics of stem cells.

[0074] In addition, FIG. 10 shows results of confirming the expression of cell surface antigen markers and osteocyte and adipocyte markers in the placenta-derived mesenchymal stem cells with enhanced expression of PRL-1, when induced to differentiate to osteocytes and adipocytes. As shown in FIG. 10, it was confirmed that when induced to differentiate to osteocytes or adipocytes, as the expression of the undifferentiation marker Oct4 decreases, the expressions of genes, such as osteocyte and adipocyte markers, i.e., osteocalsin (OC), type I collagen (Col 1), adipsin, and PPAR-r, were increased, thus demonstrating a successful differentiation to osteocytes and adipocytes. Example 3. Confirmation of Adipogenesis Inhibition

[0075] Assessment was made on whether the above-prepared placenta-derived mesenchymal stem cells with enhanced expression of PRL-1 had inhibitory effects on adipogenesis of orbital fibroblasts. In detail, orbital fibroblasts isolated from normal subjects and subjects with thyroid-associated ophthalmopathy (TAO) were placed in adipogenic induction differentiation media and cultured for 10 days, with or without the placenta-derived MSCs with enhanced expression of PRL-1 prepared above (CP-PRL-1) co-cultured therewith (for first 4 days in DMEM supplemented with 10% FBS, 33 uM biotin, 17 uM pantothenic acid, 0.2 nM T3, 10 μg/mL transferrin, 0.2 uM prostaglandin 12, 0.1 mM isobutylmethylxanthine (IBMX), 1 uM dexamethasone, and 5 ug/ml insulin/from 5-10 days w/o IBMX, dexamethaxone, insulin).

[0076] After 10 days, changes in fat accumulation in the fibroblasts were observed through Oil-Red-O staining.

[0077] The result of observation is shown in FIG. 11.

[0078] OF: Normal; TAO: Subject with thyroid associated ophthalmopathy; CP(−): Not co-cultured; CP Naive (+): Co-cultured with placenta-derived mesenchymal stem cells; CP-PRL-1 (+): Co-cultured with CP-PRL-1.

[0079] As a result, as shown in FIG. 11, it was found that fat accumulation in fibroblasts of the TAO patients, induced in differentiation media, was significantly decreased through co-culture with CP-PRL-1. Example 4. Confirmation of Ability to Regulate Adipogenesis-Associated Genes

[0080] To assess whether the placenta-derived mesenchymal stem cells with enhanced expression of PRL-1 prepared above have the ability to regulate adipogenesis-associated genes of orbital fibroblasts, orbital fibroblasts were not co-cultured with CP-PRL-1 (CP−), co-cultured with naive mesenchymal stem cells (CP Naive (+)), or co-cultured with CP-PRL-1 (CP-PRL-1 (+)), and mRNA levels of the adipogenesis-associated genes (adipsin, adiponectin, PPARγ, leptin, LPL, FABP4) expressed in these orbital fibroblasts were measured through qRT-PCR. Results thereof are shown in FIG. 12.

[0081] OF: Normal subject; TAO: Subject with thyroid associated ophthalmophathy.

[0082] As shown in FIG. 12, there was a decrease in expression level of all the adipogenesis-associated genes in the orbital fibroblasts co-cultured with the mesenchymal stem cells.

[0083] In particular, it was found that the expression levels of the adipogenesis-associated genes were significantly decreased when co-cultured with CP-PRL-1, compared to when co-cultured with naive mesenchymal stem cells.

[0084] In addition, in the above experiment groups, the gene expression levels of adipogenesis-associated factors (leptin, PPARγ) and the inflammation-associated factor TNF-α were confirmed through qRT-PCR, and their protein expression levels were confirmed by western blot. Results thereof are shown in FIG. 13.

[0085] TAO: Subject with thyroid associated ophthalmopathy. As shown in FIG. 13, it was found that when co-cultured with CP-PRL-1, there was a significant decrease in expression level of genes and proteins of adipogenesis-associated factors and inflammation-associated factors, compared to when co-cultured with naive mesenchymal stem cells. Accordingly, the placenta-derived mesenchymal stem cells with enhanced expression of PRL-1 have a desirable effect of inhibiting adipogenesis, and thus may be advantageously used as a therapeutic agent for diseases associated with adipogenesis and/or fat accumulation.