Method for preparing mesenchymal stem cell by introducing peptide, and pharmaceutical composition for treatment

Abstract

The present invention relates to: a method for preparing a mesenchymal stem cell with enhanced function by introducing a peptide; and a pharmaceutical composition using the function-enhanced mesenchymal stem cell.

Description

Method for producing mesenchymal stem cells by introducing peptides and pharmaceutical composition for therapeutic purposes

[0001] The present invention relates to a manufacturing method for enhancing the function of mesenchymal stem cells by introducing a peptide, and a pharmaceutical composition using the enhanced function mesenchymal stem cells.

[0002] Mesenchymal stem cells (MSCs) are stem cells that exist in cartilage, bone tissue, adipose tissue, and the stroma of bone marrow, differentiated from the mesoderm formed by the division of a fertilized egg. MSCs are cells that maintain stemness and self-renewal and have the ability to differentiate into various mesenchymal tissues. They can be extracted from bone marrow, adipose tissue, cord blood, synovium, bone tissue, subpatellar fat, and placenta. MSCs have immunomodulatory properties that suppress the activity and proliferation of T and B lymphocytes, inhibit the activity of natural killer cells (NK cells), and regulate the functions of dendritic cells and macrophages, making them suitable for allotransplantation and xenotransplantation. Much research is being conducted on cell therapy, focusing on the potential of MSCs.

[0003] Among the studies on mesenchymal stem cells, there are those attempting to overexpress specific factors and use them as therapeutic compositions. These include attempts to treat diseases by overexpressing PEDF in mesenchymal stem cells (Patent Document 002) and attempts to treat diseases by overexpressing PRL-1 in mesenchymal stem cells (Patent Documents 003 to 005).

[0004] PTP4A1 (protein tyrosine phosphatase type Ⅳ A1), also known as PRL-1 (phosphatase of regenerating liver-1), belongs to the group of dual protein tyrosine phosphatase enzymes that can remove phosphorylation of tyrosine residues and serine / threonine residues among 109 mammalian protein tyrosine phosphatase enzymes, and is known to affect cell growth and liver regeneration. In order to induce overexpression of PRL-1 in mesenchymal stem cells, there is a method of delivering a plasmid with the PRL-1 gene inserted into the cells using an electroporation method called AMAXA. (Patent Documents 003 to 005) However, when using electroporation, a plasmid is introduced, which may cause safety issues.

[0005] The present invention provides a method for culturing mesenchymal stem cells in a culture medium containing a peptide composed and including a core amino acid sequence of sequence number 1.

[0006] The present invention provides mesenchymal stem cells cultured in a culture medium containing a peptide composed and including a core amino acid sequence of sequence number 1.

[0007] To achieve the above task, a method for producing mesenchymal stem cells is provided, comprising the steps of: (a) providing mesenchymal stem cells; and (b) culturing the mesenchymal stem cells in a culture medium containing a PRL-1 peptide.

[0008] In the present invention, a step of culturing the mesenchymal stem cells in a culture medium that does not contain PRL-1 peptide may be further included prior to step (b).

[0009] In the present invention, after step (b), a step of culturing the mesenchymal stem cells in a culture medium that does not contain PRL-1 peptide may be further included.

[0010] In the present invention, after step (b), a step of culturing the mesenchymal stem cells in a culture medium containing PRL-1 peptide may be further included.

[0011] In the present invention, the mesenchymal stem cell may be a method for producing mesenchymal stem cells that are placental-derived mesenchymal stem cells.

[0012] In the present invention, the PRL-1 gene can be overexpressed through the PRL-1 peptide.

[0013] In the present invention, the concentration of the PRL-1 peptide may be 5 to 500 pg / ml.

[0014] In the present invention, one or more surface antigens selected from the group consisting of CD34, CD105, HLA-DR, and HLA-G can be expressed through the PRL-1 peptide.

[0015] In the present invention, the expression of Col 1 can be reduced through the PRL-1 peptide.

[0016] In the present invention, the expression of one or more genes selected from the group consisting of albumin, Cyclin D1, and HNF1a can be increased through the PRL-1 peptide.

[0017] To achieve the above task, a functionally enhanced mesenchymal stem cell is provided, which is produced by a method for producing mesenchymal stem cells, comprising the steps of (a) providing mesenchymal stem cells and (b) culturing the mesenchymal stem cells in a culture medium containing a PRL-1 peptide.

[0018] The present invention provides a composition for preventing or treating abnormal fat production and metabolic disorders, including functionally enhanced mesenchymal stem cells.

[0019] The present invention provides a composition for preventing or treating liver disease, which includes functionally enhanced mesenchymal stem cells.

[0020] According to the present invention, overexpression of PRL-1 can be induced in mesenchymal stem cells without transduction.

[0021] The functionally enhanced mesenchymal stem cells according to the present invention can be used for the prevention and treatment of abnormal fat production, metabolic disorders, liver diseases, etc.

[0022] Figure 1 shows an increase in PRL-1 gene expression in mesenchymal stem cells following introduction of PRL-1 peptide.

[0023] Figure 2 shows that the PRL-1 protein concentration in mesenchymal stem cells increases with the introduction of PRL-1 peptide.

[0024] Figure 3 shows the measurement of the doubling time of mesenchymal stem cells following introduction of PRL-1 peptide.

[0025] Figure 4 shows the measurement of PRL-1 protein concentration in mesenchymal stem cells following introduction of PRL-1 peptide.

[0026] Figure 5 compares PRL-1 gene expression in mesenchymal stem cells following introduction of PRL-1 peptide.

[0027] Figure 6 compares the amount of PRL-1 protein in mesenchymal stem cells following introduction of PRL-1 peptide.

[0028] Figure 7 compares OCT4 expression in mesenchymal stem cells following introduction of PRL-1 peptide.

[0029] Figure 8 shows the results of flow cytometry analysis in mesenchymal stem cells following introduction of PRL-1 peptide.

[0030] Figure 9 shows the results of flow cytometry analysis in mesenchymal stem cells following introduction of PRL-1 peptide.

[0031] Figure 10 shows the confirmation of osteogenic differentiation potential in mesenchymal stem cells following introduction of PRL-1 peptide.

[0032] Figure 11 shows the confirmation of fat differentiation potential in mesenchymal stem cells following introduction of PRL-1 peptide.

[0033] Figure 12 shows the confirmation of chondrogenic differentiation potential in mesenchymal stem cells following introduction of PRL-1 peptide.

[0034] Figure 13 shows the cytokine secretion ability of mesenchymal stem cells following introduction of PRL-1 peptide.

[0035] Figure 14 shows the anti-fibrotic effect of mesenchymal stem cells on hepatic stellate cell lines following introduction of PRL-1 peptide.

[0036] Figure 15 shows the effect of mesenchymal stem cells on hepatocyte regeneration following introduction of PRL-1 peptide.

[0037] The present invention can be fully achieved by the following description. It should be understood that the following description describes specific examples of the present invention and is not necessarily limited thereto. Furthermore, it should be understood that the attached drawings are provided for illustrative purposes only and are not intended to limit the present invention.

[0038] The embodiments of this disclosure are susceptible to various modifications and implementations in various forms. Therefore, it should be understood that all modifications, equivalents, and alternatives within the scope of the disclosure are acceptable, as long as they retain the spirit and technical features of the disclosure.

[0039]

[0040] Mesenchymal stem cells (MSCs) may refer to cells that maintain self-renewal and stemness maintenance and can differentiate into various mesenchymal tissues, and may include mesenchymal stem cells of mammals, for example, animals including humans. In addition, the mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cords, umbilical cord blood, bone marrow, placenta, or fat. The placental-derived mesenchymal stem cells may be derived from various tissues constituting the placenta, for example, amniotic epithelial cells, amnion, trophoblast, chorion, etc. Preferably, the placental-derived mesenchymal stem cells may be mesenchymal stem cells derived from the chorionic plate of the placenta, and more preferably, they may be mesenchymal stem cells derived from the chorionic plate membrane. The separation of mesenchymal stem cells may be performed by a method apparent to those skilled in the art.

[0041] PRL-1 (phosphatase of regenerating liver -1) may include PRL-1 derived from vertebrates, including humans, such as mammals, fish, amphibians, birds, or reptiles. In addition, the PRL-1 may also mean a precursor of PRL-1.

[0042] PRL-1 can be produced as a fusion protein. In a method for producing a fusion protein, a polynucleotide encoding PRL-1 can be linked in frame with a polynucleotide encoding another protein or peptide. Techniques known in the art can be used for this purpose. For peptides fused to PRL-1, known peptides can be used, such as FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6x His residues consisting of six histidines (His), 10x His, influenza hemagglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragments, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, alphatubulin fragment, B-tag, and Protein C fragment. Additionally, it is possible to link PRL-1 to glutathione-S-transferase (GST), influenza hemagglutinin (HA), immunoglobulin constant regions, beta-galactosidase, maltose-binding protein (MBP), or others to create fusion proteins.

[0043] As used herein, "via a PRL-1 peptide" refers to a cell being affected by the presence of a PRL-1 peptide. The process by which a cell is affected may be by the PRL-1 peptide affecting the interior of the cell.

[0044] The PRL-1 gene can be overexpressed in mesenchymal stem cells using the PRL-1 peptide. Overexpression refers to increased expression of the PRL-1 gene compared to when the PRL-1 peptide is absent.

[0045] The PRL-1 peptide can cause the mesenchymal stem cell to express one or more surface antigens selected from the group consisting of CD34, CD105, HLA-DR, and HLA-G. The PRL-1 peptide can decrease the expression of Col 1 in the mesenchymal stem cell. The PRL-1 peptide can increase the expression of one or more genes selected from the group consisting of albumin, Cyclin D1, and HNF1a in the mesenchymal stem cell.

[0046] The culture medium may include α-MEM medium or DMEM / F12 medium. The culture medium may include additional substances. Additional substances may include penicillin-streptomycin, heparin, and fibroblast growth factor.

[0047] Abnormal lipogenesis and metabolic disorders can refer to diseases caused by abnormalities in fat metabolism. Examples of abnormal lipogenesis and metabolic disorders include obesity, diabetes, dyslipidemia, metabolic disorders, hypertension, thyroid eye disease, or degenerative diseases related to abnormal lipogenesis and metabolic disorders.

[0048] Additionally, it may include metabolic-based female menopausal diseases, ovarian dysfunction (ovarian insufficiency, polycystic ovary syndrome), and metabolic osteoporosis.

[0049] Liver disease can be liver damage, liver failure, hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, liver abscess, hepatic coma, hepatic atrophy, or liver cancer. When the above liver disease persists chronically and the liver cannot perform normal function, it can be called “hepatic dysfunction.” Patients with liver dysfunction may have significantly increased expression of fibrosis-related genes compared to normal individuals; and / or significantly decreased concentrations of Cyclin D1, HNF1a, and albumin (ALB) compared to normal individuals.

[0050]

[0051] The present disclosure is described in more detail through the following examples. However, these examples merely illustrate specific embodiments of the present disclosure and are not intended to limit the present disclosure.

[0052]

[0053] 1. Manufacturing examples, examples and comparative examples

[0054] Manufacturing Example 1. Isolation and culture of placental-derived mesenchymal stem cells

[0055] Informed consent was obtained from healthy mothers who had given birth normally, and the umbilical cord was separated from the placental tissue collected during the delivery of the normal placenta. The separated tissue (chorioamniotic membranes) was placed in a 50 ml tube, and DPBS was added to remove excess blood. After scraping the upper part of the chorioamniotic membranes with a sterilized slide glass 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), the recovered floaters were collected to one side. 10 ml of enzyme solution I was added, mixed evenly, and the enzyme reaction at 37℃ was repeated twice for 15 minutes each to isolate stem cells from the tissue. The separated cell suspension was centrifuged, and the separated cells were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1 ug / ml heparin, and 25 ng / ml fibroblast growth factor-4 (FGF-4) at 37°C in an incubator with 20% O2 and 5% CO2.

[0056]

[0057] Manufacturing Example 2. Preparation of PRL-1 peptide solution

[0058] A peptide solution was prepared by adding PRL-1 and recombinant human PTP4A1 peptide (NOVUS, NBP1-50946-0.1 mg) to α-MEM medium so that the concentration of the PRL-1 peptide was 1 mg / ml. The amino acid sequence of the human PRL-1 peptide is as shown in Table 1.

[0059] SEQ ID NO: Amino acid sequence 1MGSSHHHHHHSSGLVPRGSHMARMNRPAPVEVTYKNMRFLITHNPTNATLNKFIEELKKYGVTTIVRVCEATYDTTLVEKEGIHVLDWPFDDGAPPSNQIVDDWLSLVKIKFREEPGCCIAVHCVAGLGRAPVLVALALIEGGMKYEDAVQFIRQKRRGAFNSKQLLYLEKYRPKMRLRFKDSNGHRNNC

[0060]

[0061] Example 1. Culturing mesenchymal stem cells using peptide solution (single)

[0062] (1) Passage 5 Single

[0063] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 are subcultured four times by exchanging the culture medium at intervals of 4 to 5 days. Thereafter, the peptide solution of Manufacturing Example 2 is added to the culture medium used for the fifth subculture so that the concentration of the PRL-1 peptide becomes 500 pg / ml, and cultured for 120 hours.

[0064] (2) Passage 6 Single

[0065] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 are subcultured four times by exchanging the culture medium at intervals of 4 to 5 days. Thereafter, the peptide solution of Manufacturing Example 2 is added to the culture medium used for the fifth subculture so that the concentration of the PRL-1 peptide becomes 500 pg / ml and cultured. The culture medium used for the sixth subculture is cultured without adding the peptide solution.

[0066] (3) Passage 7 Single

[0067] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 are subcultured four times by exchanging the culture medium at intervals of 4 to 5 days. Thereafter, the peptide solution of Manufacturing Example 2 of Manufacturing Example 2 is added to the culture medium used for the fifth subculture, and the cells are cultured so that the concentration of the PRL-1 peptide becomes 500 pg / ml. The culture medium used for the 6th to 7th subculture is cultured without adding the peptide solution.

[0068]

[0069] Example 2. Culturing mesenchymal stem cells using peptide solutions (multiple)

[0070] (1) passage 5 Multiple

[0071] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 are subcultured four times by exchanging the culture medium at intervals of 4 to 5 days. Thereafter, the peptide solution of Manufacturing Example 2 is added to the culture medium used for the fifth subculture so that the concentration of the PRL-1 peptide becomes 500 pg / ml and cultured.

[0072] (2) passage 6 Multiple

[0073] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 are subcultured four times by exchanging the culture medium at intervals of 4 to 5 days. Thereafter, the peptide solution of Manufacturing Example 2 is added to the culture medium used for the 5th to 6th subculture, and the cells are cultured so that the concentration of the PRL-1 peptide becomes 500 pg / ml.

[0074] (3) passage 7 Multiple

[0075] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 are subcultured four times by exchanging the culture medium at intervals of 4 to 5 days. Thereafter, the peptide solution of Manufacturing Example 2 is added to the culture medium used for the 5th to 7th subculture, and the cells are cultured so that the concentration of the PRL-1 peptide becomes 500 pg / ml.

[0076]

[0077] Comparative Example 1: Negative Control (Naive)

[0078] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 were passaged 5 to 7 times by exchanging the culture medium at intervals of 4 to 5 days and used as a negative control group.

[0079]

[0080] Comparative Example 2. Preparation of a positive control (AMAXA) using electroporation.

[0081] Mesenchymal stem cells cultured using the method of Manufacturing Example 1 are subcultured four times by exchanging the culture medium at intervals of 4 to 5 days.

[0082] Afterwards, placental chorionic valve-derived mesenchymal stem cells (CP-MSCs) were cultured in MEM-alpha medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 25 ng / ml Fibroblast Growth Factor-4 (FGF-4), and 1 ug / ml Heparin at 37°C in a CO2 incubator. The cells were washed with phosphate-buffered saline (PBS), trypsinized for 2 minutes at 37°C, and then collected with PBS and centrifuged at 1200 rpm for 5 minutes. The resulting pellet was suspended in 1 ml of culture medium and counted using a hemoglobin meter. The recovered cells were centrifuged at 200 g for 10 minutes and placed in a Nucleofector to separate the cells (5x10 5 / 100ul) was suspended and 2 ug of DNA plasmid containing PRL-1 was added. The cells were transferred to a cuvette and placed in a Nucelofection machine to start the 'U-23' program. After the program was completed, the culture dish containing new medium was quickly stabilized in a CO2 incubator at 37℃. After 4 hours, the medium in the culture dish was removed using an aspirator, and the attached cells were cultured in MEM-alpha medium containing 1.5 mg / ml neomycin (10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 25 ng / ml Fibroblast Growth Factor-4 (FGF-4), 1 ug / ml Heparin) to obtain placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene.

[0083]

[0084] 2. Analysis of manufacturing examples, examples, and comparative examples

[0085] (1) PRL-1 gene expression and protein synthesis following introduction of PRL-1 peptide

[0086] The mesenchymal stem cells of Manufacturing Example 1 were treated with 5 pg / ml, 50 pg / ml, and 500 pg / ml of the peptide solution of Manufacturing Example 2. As shown in Fig. 1, it can be confirmed that the expression level of PRL-1 mRNA increased 24 hours and 72 hours after peptide solution treatment. In addition, it can be confirmed through Western blot and protein concentration measurement of Fig. 2 that the PRL-1 protein concentration increased 24 hours and 72 hours after peptide solution treatment.

[0087] (2) Comparison of examples and comparative examples after subculture

[0088] The doubling time of cells from Examples 1 (Single), 2 (Multiple) and Comparative Example 1 (Naive) that were passaged 5 to 7 times was compared. As shown in Fig. 3, there was no significant difference compared to Naive, which was passaged without any treatment.

[0089] The concentration of PRL-1 in Example 1 (Single), 2 (Multiple) and Comparative Example 1 (Naive) that were passaged 5 to 7 times, and Comparative Example 2 (AMAXA) using electroporation were analyzed by ELISA. As shown in Fig. 4, the synthesis of PRL-1 protein was confirmed.

[0090] The PRL-1 mRNA expression levels and protein amounts of Examples 1 (Single), 2 (Multiple), Comparative Example 1 (Naive), and Comparative Example 2 (AMAXA) that were subcultured 5 to 7 times were compared. As shown in Fig. 5, it can be seen that the PRL-1 mRNA expression levels of Examples 1 and 2 were low when subcultured 6 to 7 times, but as can be seen in Fig. 6, there was no significant difference in the protein amount of PRL-1.

[0091] The expression levels of the undifferentiated marker OCT4 in Examples 1 (Single), 2 (Multiple) and Comparative Example 1 (Naive) that were passaged 5 to 7 times and Comparative Example 2 (AMAXA) that was passaged 9 to 11 times were compared, and can be confirmed in Figure 7.

[0092] The results of flow cytometry (FACS) measurements of Examples 1 (Single), 2 (Multiple) and Comparative Example 1 (Naive) passaged 5 to 7 times can be confirmed in FIGS. 8 and 9. Through FIGS. 8 and 9, it was confirmed that the results were negative for hematopoietic markers (CD34 and HLA-DR) (FIGS. 2A and 2B) and positive for non-hematopoietic markers (CD13, CD90, CD105, HLA-ABC and HLA-G). In addition, it can be seen that Examples 1 and 2 do not differ significantly from Comparative Example 1.

[0093] (3) Confirmation of differentiation ability of examples and comparative examples

[0094] To confirm the differentiation ability as mesenchymal stem cells, differentiation of Examples 1 and 2 was induced.

[0095] To confirm the osteogenic potential of Examples 1 and 2, this was confirmed through von Kossa staining, which stains for mineralization of calcium, potassium, etc. in tissues. As shown in Fig. 10, staining was confirmed in the Examples and Comparative Examples. In addition, as shown in Fig. 10, the osteogenic potential can be confirmed through the increased expression of the bone cell-specific gene (Osteoclcin) in the Examples and Comparative Examples compared to undifferentiated cells (Undiff).

[0096] To confirm the adipogenic potential of Examples 1 and 2, this was confirmed through Oil Red O staining, which stains lipids. As shown in Figure 11, staining was confirmed in the Examples and Comparative Examples, and the difference in values ​​was not significant. In addition, as shown in Figure 11, the expression of the adipocyte-specific gene (Adipsin) in the Examples and Comparative Examples increased compared to undifferentiated cells, confirming the adipogenic potential.

[0097] In order to confirm the chondrogenic potential of Examples 1 and 2, this was confirmed through Alcian blue staining, which stains cartilage. As can be seen in Fig. 12, when the expression levels of Sox9 and ColⅡ genes related to chondrogenic potential in Examples 1, 2 and Comparative Example 2 are compared with undifferentiated cells (Undiff), it can be confirmed that the chondrogenic potential is low. However, it can be confirmed that the expression levels of Sox9 and ColⅡ genes in Example 2 are higher than in Example 1 and Comparative Example 2.

[0098] (4) Confirmation of cytokine secretion ability of examples and comparative examples

[0099] To confirm the cytokine secretion ability of placental-derived mesenchymal stem cells following introduction of PRL-1 peptide, the cytokine secretion ability of Comparative Example 1 (Naive), Examples 1 (Single) and 2 (Multiple) was compared.

[0100] Comparative Example 1, Examples 1 and 2, which were subcultured 7 times, were cultured for 24 hours, then replaced with a medium excluding FBS and cultured for 24 hours. Thereafter, the cytokine secretion ability was confirmed using the medium. In Fig. 13, it was confirmed that the expression of factors involved in regenerative ability (growth factor), immune-related factors, factors involved in metabolic activity, and factors involved in vascular regeneration in placental-derived mesenchymal stem cells increased compared to naïve following the introduction of PRL-1 peptide. It can be confirmed that Example 1 secreted a higher amount of cytokines than Example 2.

[0101] (5) Confirmation of anti-fibrotic effect in hepatic stellate cell lines

[0102] Hepatic stellate cell lines were cultured in T-HSC medium for 24 hours, and then treated with 2 ng / ml TGF-b to induce fibrosis for 24 hours. Thereafter, the cells were treated with Example 1, Example 2, Comparative Examples 1 and 2, and cultured for 24 hours to confirm the antifibrotic effect. The antifibrotic effect can be confirmed through the expression of Col 1 and the amount of Col 1 protein in Fig. 14.

[0103] (6) Confirmation of regenerative effect in liver cells

[0104] Hepatocyte cell lines were cultured in WB-F344 medium for 24 hours, and then chemically treated (LCA) to induce cell damage for 48 hours. Thereafter, the cells were treated with Examples 1, 2, and Comparative Examples 1 and 2, and cultured for 24 hours to confirm the cell regeneration effect. As shown in Fig. 15, the regeneration effect of hepatocytes can be confirmed through the increased expression of albumin (ALB), Cyclin D1, and HNF1a, which are secreted by normal hepatocytes.

Claims

1. (a) A step of providing mesenchymal stem cells; (b) A method for producing mesenchymal stem cells, comprising a step of culturing the mesenchymal stem cells in a culture medium containing PRL-1 peptide.

2. In paragraph 1, (b) A method for producing mesenchymal stem cells, further comprising a step of culturing the mesenchymal stem cells in a culture medium that does not contain PRL-1 peptide prior to step (b).

3. In paragraph 1 or 2, (b) A method for producing mesenchymal stem cells, further comprising a step of culturing the mesenchymal stem cells in a culture medium that does not contain PRL-1 peptide after step (b).

4. In paragraph 1 or 2, (b) A method for producing mesenchymal stem cells, further comprising a step of culturing the mesenchymal stem cells in a culture medium containing PRL-1 peptide after step (b).

5. In paragraph 1, The above mesenchymal stem cells are a method for producing mesenchymal stem cells that are placental-derived mesenchymal stem cells.

6. In paragraph 1, A method for producing mesenchymal stem cells that overexpress the PRL-1 gene through a peptide containing the above PRL-1 core amino acid sequence.

7. In paragraph 1, A method for producing mesenchymal stem cells, wherein the concentration of the PRL-1 peptide in the culture medium is 1 to 1000 pg / ml.

8. In paragraph 1, A method for producing mesenchymal stem cells that express one or more surface antigens selected from the group consisting of CD34, CD105, HLA-DR, and HLA-G through the above PRL-1 peptide. 9.(a) Step of providing mesenchymal stem cells; (b) Functionally enhanced mesenchymal stem cells produced by a method for producing mesenchymal stem cells, comprising the step of culturing the mesenchymal stem cells in a culture medium containing a peptide including a PRL-1 core amino acid.

10. In paragraph 9, A composition for preventing or treating abnormal fat production, metabolic disorders and degenerative diseases, comprising the above-mentioned functionally enhanced mesenchymal stem cells.

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