KR101892071B1 - Process for preparing immortalized mesenchymal stem cell line - Google Patents

Abstract

The present invention provides a method for producing an immortalized mesenchymal stem cell line, which comprises inducing overexpression of a pituitary tumor transforming gene 1 (PTTG1). In addition, the present invention provides an immortalized placenta-derived mesenchymal stem cell line obtained by the above production method.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for preparing an immortalized mesenchymal stem cell line,

The present invention relates to a method for producing an immortalized mesenchymal stem cell line, and more particularly to a method for producing an immortalized mesenchymal stem cell line, which comprises inducing overexpression of a pituitary tumor transforming gene 1 (PTTG1) And a manufacturing method thereof. Further, the present invention relates to an immortalized mesenchymal stem cell line obtained by the above-mentioned production method.

Human mesenchymal stem cells (hMSCs) can be isolated from a variety of organs, have self-renewal capacity and are also capable of differentiating into multiple lineages under specific culture conditions And thus it is expected to be used in regenerative medicine. Although MSCs have autonomous proliferative capacity, in general, mesenchymal stem cells have limited self-proliferative activity and also have an average life span of less than 20% (population doublings), so conducting studies for potential clinical use It is difficult. Normal human cells undergo limited cell division during culture prior to entering a non-cleavage state known as "senescence" (Campisi et al. (1996) Exp Gerontol 31: 7-12). Replicative senescence, first described by Hayflick (Hayflick L (1965) Exp Cell Res 37: 614-636), has been shown to induce arrested growth and degeneration in normal somatic cells following a limited number of cell divisions It is the state of an altered function.

Recently, human placenta-derived MSCs (PDSCs) have been identified as alternatives to traditional MSCs such as bone marrow-derived MSCs (BM-MSCs). PDSCs have the advantages of self-proliferative activity and differentiation into various systems, and are also readily available, ethically problematic, and have strong immunomodulatory effects (Igura et al. (2004) Cytotherapy 6: 543-553). The present inventors have identified several kinds of PDSCs in the normal placenta based on the placental structure (Kim et al. (2011) Cell Tissue Res 346: 53-64). Proliferative activity of chorionic-plate-derived MSCs (CP-MSCs) and warton jelly-derived MSCs (Whaton's gel-derived MSCs, WJ-MSCs) were higher than those of other types of PDSCs. Although the aut-proliferative activity is higher than conventional MSCs, the average lifespan of CP-MSC and WJ-MSC was population doublings, which limits the ability to obtain enough cells for use in clinical applications. Thus, the limited autogenous activity of mesenchymal stem cells is the biggest obstacle to the application of stem cell therapy in regenerative medicine.

Senescent cells are arrested at the G1 stage of the cell cycle and exhibit a large, flattened form. Although senescent cells are metabolically active, they have limitations for cell proliferation through DNA synthesis even for growth factor stimulation (Fridman et al. (2006) J Gerontol A Biol Sci Med Sci 61: 879-889 ). This is because these cells have one or more short-acting dysfunctional telomeres due to persistent subculture and also lack telomerase expression (Harley et al. (1990) Nature 345: 458-460). Cell cycle-related factors have been reported to be closely related to the autoproliferative activity of MSCs (Napolitano et al. (2007) J Cell Sci 120: 2904-2911). In addition, a strong link between telomerase expression and many types of cancer has been observed (Miura et al. (2006) Cancer Sci 97: 1366-1373). Primary cells isolated from each tissue, Can be induced to infinitely grow in vitro through a process known as ex vivo immortalization by genetic manipulation using viruses and the like (Okamoto et al. (2002) Biochem Biophys Res Commun 295: 354- However, the existing methods for expanding adult stem cells to obtain a large number of cells are limited, and problems including safety for clinical application remain.

In contrast, pituitary tumor transforming gene 1 (PTTG1), also known as securin, was isolated from rat pituitary tumor cells in 1997 (Pei and Melmed (1997) Mol Endocrinol 11: 433-441 ) And regulates sister chromatid separation during mitosis (Zou et al. (1999) Science 285: 418-422). PTTG1 is overexpressed in rapidly proliferating cells and a variety of early tumor and tumor cell lines and has been implicated in cell replication, cell cycle-progression, and proper cell division (Heaney et al. (2000) Lancet 355: 716-719; Saez C, et al. 1999) Oncogene 18: 5473-5476; Tong Y, et al. (2007) Oncogene 26: 5596-5605; Wang Z et al. 2001 Mol Endocrinol 15: 1870-1879), chromosome stability (Jallepalli et al. (2001) Cell 105: 445-457), and cellular senescence of cancer cells (Chesnokova et al. (2007) Cancer Res 67: 10564-10572). PTTG1 expression levels in cancer cells are associated with tumor invasion, recurrence, and poor prognosis, and PTTG1 has been identified as a key gene associated with tumor metastasis (Ramaswamy et al. (2003) Nat Genet 33: 49- 54).

The present invention has been extensively studied to establish an immortalized mesenchymal stem cell line that overcomes the limited autogenous activity of mesenchymal stem cells. Surprisingly, it has been found that when overexpressed in mesenchymal stem cells, PTTG1 (gene of SEQ ID NO: 1), which is overexpressed in tumor cells and is known to be involved in tumor invasion, recurrence, and metastasis, mesenchymal stem cells can be immortalized.

Accordingly, it is an object of the present invention to provide a method for producing an immortalized mesenchymal stem cell line, which comprises inducing overexpression of PTTG1 (gene of SEQ ID NO: 1).

It is another object of the present invention to provide an immortalized mesenchymal stem cell line obtained by the above production method.

According to one aspect of the present invention, there is provided a method of producing a stem cell comprising: (a) providing mesenchymal stem cells; And (b) inducing overexpression of the gene of SEQ ID NO: 1 in the mesenchymal stem cell.

The mesenchymal stem cells of step (a) may be placenta-derived mesenchymal stem cells and may be, for example, mesenchymal stem cells derived from a chorionic plate, amnion, or waton jelly have. In one embodiment, the placenta-derived mesenchymal stem cells are mesenchymal stem cells derived from the chorionic plate. Step (b) can be carried out by isolating the cells overexpressing the PTTG1 gene from the cells obtained by transfecting the mesenchymal stem cells with the gene of SEQ ID NO: 2. In addition, the transfection can be preferably carried out by electroporation.

According to another aspect of the present invention, an immortalized placenta-derived mesenchymal stem cell line overexpressing the gene of SEQ ID NO: 1 is provided.

The immortalized placenta-derived mesenchymal stem cell can be an immortalized placenta-derived mesenchymal stem cell strain derived from a chorionic plate, amniotic membrane, or waton jelly overexpressing the gene of SEQ ID NO: 1. In one embodiment, the immortalized placenta-derived mesenchymal stem cell line is KCLRF-BP-00339.

It has been revealed by the present invention that when PTTG1 (the gene of SEQ ID NO: 1) is overexpressed in mesenchymal stem cells, mesenchymal stem cells can be immortalized. The immortalized mesenchymal stem cell line obtained according to the present invention maintains the stemness capacity and multipotency of native MSCs. For the immortalization of MSCs, it has been found by the present invention that the interaction of PTTG1 with the TERT gene (the gene of SEQ ID NO: 2) is required. That is, immortalization of MSCs requires TERT bound to PTTG1 to form complexes with chaperones such as Ku70 and heat shock protein 90 (HSP90), and is immortalized through the interaction balance between PTTG1 and autonomic It has been found by the present invention that the mesenchymal stem cell line exhibits differentiation potential and also promotes auto-proliferation. Thus, the immortalized mesenchymal stem cell line obtained according to the present invention can be useful for the application of stem cell therapy in regenerative medicine.

Brief Description of the Drawings Fig. 1 is a graph showing the results of immunoprecipitation of native placenta-derived mesenchymal stem cells (PDSCs) and the immortalized mesenchymal stem cell line KCLRF-BP-00339 (hTERT-PDMSCs) obtained from PDSCs through transfection of the TERT gene and overexpression of the PTTG1 gene )) (TERT + cells). Figure 1a shows the form of TERT + cells altered by immortalization (Bars: 50 [mu] m). Figure IB shows in vitro telomerase activity of native PDSCs and TERT + cells. Figure 1C shows the results of RT-PCR analysis of stem cell markers. Figure 1d shows the immunophenotype of hematopoietic and mesenchymal stem cells of native PDSCs and TERT + cells by flowcytometry. Figure 1e shows the immunophenotype of HLA-ABC, DR, and G of native PDSCs and TERT + cells by flow cytometry. Percentages of cells positive for various cell surface markers were calculated by comparing the isotype controls with the target protein signal. FIG. 1F shows the result of cell cycle analysis by FACS (fluorescence-activated cell sorter) scan. Figure 1g shows the growth curves of native PDSCs and TERT + cells.
Figure 2 shows the differentiation of native PDSCs and TERT + cells into adiopogenic lineages and hepatogenic lineages. Figure 2a shows the accumulation of oil droplets measured by oil-red O staining in differentiated native PDSCs and TERT + cells. Cells were counter-stained with hematoxylin. Bars are 50 μm. Figure 2b shows the adipocyte differentiation of naturally occurring PDSCs and TERT + cells, as confirmed by mRNA detection, which is an adipocyte marker, lipofectamine lipase and PPAR-y. Figure 2c shows hepatocyte differentiation of native PDSCs and TERT + cells revealed by the uptake of indocyanine green. Bars are 50 μm. Figure 2d shows hepatocyte differentiation of native PDSCs and TERT + cells identified by RT-PCR (AFP, albumin, and TAT). β-Actin was used as an internal control.
FIG. 3 shows the results of analysis of expression of auto-proliferation, cell cycle, antiapoptotic proteins and autophagic proteins in native PDSCs and TERT + cells. Figure 3a shows the expression of similar or increased self-proliferating markers in TERT + cells relative to native PDSCs. Figure 3B shows the increase of cell cycle-related protein and the decrease of cell cycle regulator in TERT + cells. Figure 3c shows the increase of anti-apoptotic protein Bcl-2 and the decrease of apoptotic protein in TERT + cells. Figure 3d shows the increase of autophagic proteins phosphorylated-mTOR and LC3 in TERT + cells. D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. FIG. 3E shows that when TERT + is induced in native PDSCs (Naive) cells, the expression of TERT gene is about three times higher than that of Naive cells, whereas the expression of PTTG1 is about four times higher than that of Naive cells.
Figure 4 shows the effect of PTTGl expression on cell viability and apoptosis in native PDSCs and TERT + cells. Figure 4a shows the decrease in expression of PTTG1 and PTTG2 in cells treated with siRNA-PTTG1, which were evaluated for RT-PCR analysis. 4B shows the result of evaluating telomerase activity through TRAP analysis of cells in which PTTG1 expression is decreased. FIG. 4C shows the expression decrease of PTTG1, TERT, Oct-4, and Ku70 by down-regulation of PTTG1. The expression of heat shock protein 90 (HSP90) did not change. Figure 4d shows a decrease in cell cycle factor by knock-down of PTTG1 and an increase in p21. FIG. 4E shows the ratio of anti-apoptotic proteins in the cells treated with siPTTG1. The apoptotic protein was increased in cells treated with siPTTG1. Figure 4f shows a decrease in autophagic activity after siPTTG1 treatment.
Figure 5 shows that PTTG1 reduction induces cell senescence. Figure 5a shows the activity (blue) of cell senescence-associated [beta] -galactosidase at pH 6.0 used as a marker of replication senescence. Bars are 50 μm. 5B shows the results of cell cycle analysis by FACScan after siPTTG1 treatment. FIG. 5c shows the results of cell death analysis using a 3/7 enzyme-linked immunosorbent assay (ELISA).
Figure 6 shows that the interaction of HSP90 and Ku70 with TERT and PTTG1 is dependent on PTTG1 expression. Figure 6a shows the interaction of PTTG1, HSP90, and Ku70 with TERT revealed by immunoprecipitation (IP) using an anti-TT antibody. Figure 6b shows the interaction of Ku70 with TERT, PTTG1, and HSP90 as measured by immunoprecipitation with anti-Ku70 antibody.
Figure 7 shows the regulation mechanism of TERT and PTTG1 balance through association with Ku70. PTTG1 is overexpressed in TERT + cells, and PTTG1 interacts with TERT through Ku70. Changes in PTTG1 expression control the physiology of cells including autologous proliferation, apoptosis, and cell senescence through the binding of PTTG1 to Ku70 in TERT + cells.
Figure 8 shows the karyotypic results of native PDSCs and TERT + cells.
Figure 9 shows that HSP70 is expressed and interacts with TERT and Ku70 independent of PTTG1 expression in native PDSCs and TERT + cells.

(A) providing mesenchymal stem cells; And (b) inducing overexpression of the gene of SEQ ID NO: 1 in the mesenchymal stem cell. The present invention also provides a method for producing an immortalized mesenchymal stem cell line.

The present inventors have found that when PTTG1 (gene of SEQ ID NO: 1) is overexpressed in mesenchymal stem cells, mesenchymal stem cells can be immortalized. The immortalized mesenchymal stem cell line obtained according to the present invention maintains the stemness capacity and multipotency of native MSCs.

Step (a) can be carried out by separating mesenchymal stem cells through conventional biological and / or genetic engineering methods from tissues, organs, of mammals, including humans, isolated in vitro. For example, the mesenchymal stem cells may be cells obtained from bone marrow, fat, placenta, etc. Preferably, the mesenchymal stem cells are placenta-derived mesenchymal stem cells, and the mesenchymal stem cells derived from the placental tissue, such as chorionic plate, amnion, warton jelly, etc., Lt; / RTI > The placenta-derived stem cells can be obtained by known methods (for example, Current Protocols in Stem Cell Biology 1E.3.1-1E.3.10 and 1E.5.1-1E5.11). In one embodiment, the placenta-derived mesenchymal stem cells are mesenchymal stem cells derived from the chorionic plate.

Step (b) can be performed by inducing overexpression of PTTG1 according to conventional genetic engineering methods. For example, cells transduced with overexpression of PTTG1 can be isolated by transforming the human TERT gene (the gene of SEQ ID NO: 2) by electroporation method and then separating cuboidal cells.

On the other hand, it has been found by the present invention that the interaction of PTTG1 with the TERT gene (the gene of SEQ ID NO: 2) is required for the immortalization of MSCs. That is, immortalization of MSCs requires TERT bound to PTTG1 to form complexes with chaperones such as Ku70 and heat shock protein 90 (HSP90), and is immortalized through the interaction balance between PTTG1 and autonomic It has been found by the present invention that the mesenchymal stem cell line exhibits differentiation potential and also promotes auto-proliferation.

Thus, in step (b), the mesenchymal stem cell line can be obtained by isolating the cell overexpressing the PTTG1 gene from the cell obtained by transfection of the mesenchymal stem cell with the gene of SEQ ID NO: 2. The transfection may be carried out using a suitable gene delivery system, for example, an appropriate expression vector or viral vector comprising the gene of SEQ ID NO: 2. Preferably, the transfection is carried out by electroporation, which can avoid safety problems associated with noeplastic transformation. The electroporation may be performed using, for example, the AMAXA gene delivery system.

The present invention also provides an immortalized placenta-derived mesenchymal stem cell line produced by the above method. That is, the present invention provides an immortalized placenta-derived mesenchymal stem cell line overexpressing the gene of SEQ ID NO: 1.

The immortalized placenta-derived mesenchymal stem cell line may be an immortalized placenta-derived mesenchymal stem cell line derived from placental tissue such as chorionic plate, amniotic membrane, warton jelly, etc. overexpressing the gene of SEQ ID NO: 1. In one embodiment, the immortalized placenta-derived mesenchymal stem cell line is deposited under accession number KCLRF-BP-00339 deposited on March 24, 2015 by the Korean Cell Line Research Foundation (KCLRF) .

Hereinafter, the present invention will be described in more detail with reference to concrete test examples. However, these test examples are intended to illustrate the present invention only, and the scope of the present invention is not limited to these test examples.

1. Materials and Test Methods

(1) Cell culture

The normal placenta obtained when pregnancy was not medically, obstetrical, and surgical complications and was delivered at gestation over 37 weeks was used. All women who contributed to this study provided written informed consent prior to sampling. The use of this sample for research purposes has been approved by the Institutional Review Board of CHA General Hospital, Seoul, Korea. Natural PDSCs were harvested according to reported reports (Lee MJ, et al. J Cell Biochem (2010) 111: 1453-1463). Namely, amniotic membrane and innermost membrane were removed from chorionic membrane and decidual membrane of neglected person. Cells scraped from the membranes were treated with 0.5% collagenase IV (Sigma, St. Louis, Mo., USA) for 30 minutes at 37 ° C. The harvested cells were cultured in a T25 flask (BD Biosciences, San Jose, Calif.) At 2 x 10 ⁵ cells / cm ² with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin Island, NY, USA) supplemented with Ham's F-12 / DMEM. All cell culture medium was supplemented with 100 μg / ml penicillin, 100 μg / ml streptomycin, 25 ng / ml FGF4 (Peprotech Inc., NJ, USA), 1 μg / ml heparin (SIGMA) (GIBCO-BRL) supplemented with fetal calf serum (GIBCO-BRL).

(2) Isolation of hTERT gene transfected and immortalized cells

Natural PDSCs (6 x 10 ⁴ cells / cm ² ) were harvested and transfected with the hTERT gene using the AMAXA system using a human MSC nucleofactor kit (Lonza Ltd, NJ, USA) . After hTERT gene was electroporated into native PDSCs, the medium was changed once every two days and the changes in cell morphology were observed. The hTERT gene-overexpressing cells were maintained on hTERT gene expressing cells for 3 months with G418 (200 ug / ml) -containing medium, and only hTERT gene-introduced cells were selected. The morphology of the obtained immortalized cells (hereinafter, referred to as 'TERT + cells') was transformed into a cuboidal form in a spindle form of a typical mesenchymal stem cell, and immunocytochemistry Overexpression of hTERT gene and PTTG1 overexpression was confirmed. Immortalized cells obtained were deposited with the Korean Cell Line Research Foundation (KCLRF, Korean Cell Line Research Foundation) and received grant number KCLRF-BP-00339 on March 24, 2015.

(3) RT-PCR analysis

Total RNA was extracted from cells growing on 6-well plates using RNeasy plus mini kits (QIAgen, Valencia, CA, USA). CDNA was synthesized by reverse transcription (RT) from total RNA (1 μg) using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Polymerase chain reaction (PCR) amplification was performed in a 25-μl reaction containing 100 ng cDNA template, 2.5 U / μl DNA polymerase (Solgent, South Korea), and 200 μM dNTP, 1). beta -actin was used as an internal control. The cDNA was amplified to 35 cycles (20 sec at 95 캜, 40 sec at the appropriate annealing temperature shown in Table 1, and 1 min at 72 캜). The amplified PCR product was electrophoresed on a 1.2% agarose gel containing 1.5 μg / ml ethidium bromide and visualized under UV light.

Marker gene sense/
Antisense The sequence (5 '- > 3') order
number Size (bp) Temperature
(° C) Diabetic Oct-4 sense AGT GAG AGG CAA CCT GGA GA 3 273 54 Antisense GTG AAG TGA GGG CTC CCATA 4 Nanog sense TTC TTG ACT GGG ACC TTG TC 5 300 54 Antisense GCT TGC CTT GCT TTG AAGCA 6 Sox-2 sense AGA ACC CCA AGATGC ACA AC 7 200 52 Antisense GGG CAG CGT GTA CTT ATC CT 8 NF-68 sense GAG TGA AAT GGC ACG ATA CCTA 9 700 58 Antisense TTT CCT CTC CTT CTT CTT CAC CTT C 10 Cardiac sense GGA GTT ATG GTG GGT ATG GGT C 11 500 58 Antisense AGT GGT GAC AAA GGA GTA GCCA 12 AFP sense ATG CTG CAA ACT GAC CAC GC 13 500 55 Antisense GCT TCG CTT TGC CAATGC TT 14 HLA-G sense GCG GCT ACTACA ACC AGA GC 15 900 58 Antisense GCA CAT GGC ACG TGT ATC TC 16 Adipocytic Lipoprotein lipase sense GTC CGT GGC TAC CTG TCAT 17 717 59 Antisense AGC CCT TTC TCA AAG GCT TC 18 PPAR-R sense GGA AAG ACA ACA AAC AAATCA C 19 414 59 Antisense TGC ATT GAA CTT CAC AGC AAA C 20 Liver cell AFP sense ATG CTG CAA ACT GAC CAC GC 21 200 55 Antisense GCT TCG CTT TGC CAATGC TT 22 albumin sense CCC CAA GTG TCA ACT CCA AC 23 450 55 Antisense CCA GAA GAC ACC CTC CAA AGG AC 24 TAT sense AAC GAT GTG GAG TTC ACG G 25 288 59 Antisense GAC ACATCC TCA GGA GAATGG 26 PTTG series PTTG1 sense AAG GAA AAT GGA GAACA GGC 27 237 55 Antisense GCT TGG CTG TTT TTG TTT GAG G 28 PTTG2 sense CTG ATC TAC GTT GAT AAG GAA 29 334 55 Antisense CTATTT CTG GAT AGG CGT CAT CTG 30 inside
Control group beta -actin sense TCC TTC TGC ATC CTG TCA GCA 31 300 58 Antisense CAG GAG ATG GCC ACT GCC GCA 32

(4) Cell growth and cell cycle analysis

To analyze the cell cycle, a fluorescence-activated cell sorter (FACS) assay was performed on native PDSCs and TERT + cells (1 x ¹⁰⁶ cells). After harvesting, the cells were fixed with 70% ethanol at room temperature for 10 minutes. RNase (0.5 μg / ml) and propidium iodide (50 μg / ml) were added to the cells, and the cells were incubated at 37 ° C for 30 minutes. The treated cells were analyzed with a BD FACS Vantage ^™ SE Cell Sorter equipped with Becton and Dickson ModiFit LT software (BD Bioscience).

(5) FACS analysis

To detect cell-surface antigens, native PDSCs and TERT + cells were harvested using TrypLE Express with phenol red (GIBCO-BRL). The harvested cells were suspended in PBS containing either mouse immunoglobulin G or a specific concentration of fluorescein isothiocyanate (FITC) - or phycoerythrin (PE) -conjugated antibody (Table 2) And incubated in 100 [mu] l of buffer for 30 minutes at room temperature. Samples were analyzed with BD FACSVantage ^TM SE Cell sorter (Becton & Dickinson). Results were analyzed with Cell-Quest software (Becton & Dickinson).

company Bell CD34-PE BD mouse CD45-FITC BD mouse CD90-PE BD mouse CD105-FITC R & D mouse HLA-ABC-FITC BD mouse HLA-DR-FITC BD mouse HLA-G-FITC Abcam mouse IgG-FITC Souther Biotech mouse IgG-PE BD mouse

(6) Differentiation of natural and immortalized PDSCs into adipogenic lineages and hepatogenic lineages

Adipogenic differentiation: To induce differentiation into adipocytes, native PDSCs and TERT + cells were treated with 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine, at a density of 5 × 10 ³ cells / cm ² . IBMX), 0.2 mM indomethacin, 1.7 [mu] M insulin, 10% FBS, and 1 x penicillin-streptomycin. The medium for fat cell differentiation maintenance was changed twice a week. After 21 days, the cells were fixed in 4% PFA and incubated with Oil Red O (Sigma-Aldrich) for 1 hour to stain lipids to visualize lipid vesicles. Nuclei were counter-stained with Mayer's hematoxylin (Sigma-Aldrich) for 1 minute

Hepatogenic differentiation: To induce the differentiation of native PDSCs and TERT + cells into hepatocytes, cells were seeded at a density of 2 x 10 ⁴ cells / cm ² with 20 ng / ml EGF and 10 ng / ml bFGF Was added to the basal medium (60% DMEM-LG, 40% MCDB201, 2% FBS, 1% penicillin-streptomycin) and grown to 60% confluence. When the cells were 70-80% confluent, the culture medium was replaced with an early inducing medium to differentiate into hepatocytes. For initiation, cells were incubated for 7 days in basal medium supplemented with 10 ng / ml BMP4, 20 ng / ml HGF, 10 ng / ml bFGF, and 0.61 g / L nicotinamide. For progression to the maturation step, the cells were incubated with 2% FBS, 1 μmol / L dexamethasone, 1 × ITS + premix, and 20 ng / ml oncostatin M (OSM) Lt; RTI ID = 0.0 > supplemented < / RTI > After 7 days of incubation in the terminal medium, hepatocyte differentiation was confirmed by ICG uptake by hepatogenic induced cells. Subsequently, hepatocyte-derived cells were harvested and analyzed for hepatocyte-specific gene expression by RT-PCR.

(7) Western blotting

Cells were washed with PBS and lysed in 100 μl of cold RIPA buffer (SIGMA) using a protease inhibitor cocktail (Roche, Mannheim, Germany). Cell lysates were centrifuged at 13000 x g for 15 minutes at 4 ° C. The supernatant was harvested and its protein concentration was measured with a BCA protein assay kit (Pierce, Rockford, IL, USA). For electrophoresis, 40 μg of protein was dissolved in sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue) , Boiled at 90 ° C for 5 minutes, and then separated on an 8% SDS reducing gel. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-rad, Hercules, CA, USA) using a trans-blot system (Bio-rad). The blots were blocked for 1 hour at room temperature in phosphate-buffered saline (PBS) containing 8% non-fat dry milk (BD, Sparks, MD, USA) and 0.05% And incubated overnight at 4 [deg.] C in PBS-T containing 2% non-fat dry milk with secondary antibody (Table 3). The blots were incubated for 1 hour with PBS-T containing 2% non-fat dry milk along with horseradish peroxidase-conjugated secondary antibodies (1: 10000 dilution; Bio-rad) ≪ / RTI > at room temperature. Proteins were then visualized with an ECL detection system (Amersham, Piscataway, NJ, USA).

Marker gene company Bell Size (kDa) Self-proliferation TERT Rackland rabbit 120 Oct-4 Chemicon mouse 44 Nanog Cell Signaling rabbit 42 SCF santacruz rabbit 45 Heat shock protein
(Heat-shock protein)
HSP90 BD mouse 90 HSP70 Stress Marq mouse 70 Cell cycle
(Cell cycle) Cdk-4 Invitrogen mouse 43 p53 Santacruz mouse 53 p21 Abcam rabbit 21 Cyclin E1 Abfrontier rabbit 47 PTTG1 Invitrogen rabbit 28 PTTG2 Abnova mouse 21 Cell death
(Apoptosis)
PARP 셀 신호링 rabbit Full: 116
Cleavage: 89 Bad Santacruz mouse 25 Bax Santacruz rabbit 23 Bcl-2 Santacruz rabbit 26 Caspase-9 Abcam rabbit 43 Active caspase-3 BD rabbit 17 & 20 Self-predation
(Autophagy)
Phospho-mTOR 셀 신호링 rabbit 289 LC3 Novus rabbit 17 & 19 Atg5-12 Santacruz mouse Atg 12: 21
Atg12-APG5 conjugate: 60 Internal control GAPDH Abfrontier rabbit 37 Immune precipitation TERT Santacruz rabbit 120 Ku70 Thermo mouse 70

(8) Immunoprecipitation

Cells were scraped from the culture dish using 200 [mu] l of cold RIPA buffer (SIGMA) containing protease inhibitor cocktail (Roche). Cell lysates were centrifuged at 13000 x g for 15 minutes at 4 ° C. The supernatant was harvested and the protein concentration was measured with a BCA protein assay kit. For immunoprecipitation, 300 μg of protein in RIPA buffer was incubated with 2 μg of antibody per 300 μg of total protein for 3-4 hours at 4 ° C. The pre-mixture of protein and antibody was then incubated with 20 [mu] l of a 50% slurry of protein G-agarose beads (50% slurry of protein G-agarose beads, GenDEPOT) overnight at 4 [deg.] C with constant stirring. After centrifugation at 1000 rpm for 1 minute at 4 ° C, the supernatant was discarded and the agarose-bead pellet was washed with PBS. After the final wash, the agarose-bead pellet was resuspended in 20 μl of sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% Bromophenol Blue) and boiled at 90 < 0 > C for 5 minutes. The agarose beads were then removed by centrifugation and the supernatant immediately loaded onto an 8% SDS reducing gel. Western blotting was then carried out as described above.

(9) Indocyanine green uptake analysis

The cultured cells were washed with PBS and then indocyanine green (ICG) solution (100 mg / ml) was added to the plate at a final concentration of 1 mg / ml and incubated at 37 ° C for 90 minutes. The plates were then washed with PBS and backfilled with DMEM containing 10% FBS. ICG was completely removed from the cells after 72 hours.

(10) TRAP analysis

Induce specific amplification of telomerase-mediated elongation products; Telomerase activity was measured using a PCR-based technique using the modified telomerase repeat amplification protocol (TRAP), which allows visualization of these products by ethidium bromide staining. This technique specifically amplifies the telomerase-mediated elongation product under the following conditions: denaturation at 90 ° C for 3 minutes, followed by denaturation at 94 ° C for 45 seconds, annealing at 53 ° C for 45 seconds, , And one cycle of extension at 72 캜 for 31 cycles. The product was loaded onto a polyacrylamide gel. After electrophoresis, the gel was stained with SYBR Green I. The primer sequences are: TS primer 5'-AATCCGTCGAGCAGAGTT-3 '(SEQ ID NO: 33); CX primer 5'-CCCTTACCCTTACCCTTACCCTAA-3 '(SEQ ID NO: 34).

(11) Transfection of siRNA-PTTG1

For PTTG1 knock-down, 5 x ¹⁰⁶ cells were placed in 10-cm culture dishes and cultured at 37 ° C in 5% CO ₂ . After 24 hours, the medium was removed from the culture dish and 4 ml of OPTI-MEM was added. In the following step, Lipofectamine 2000 (Invitrogen) protocol was performed. Cells were transfected with 100 nM siRNA-PTTG1 to down-regulate the PTTG1 gene and incubated for 48 hours. Finally, the cells were harvested for analysis.

(12) Staining with SA-β-galactosidase

Cells were washed with PBS, fixed in 4% formaldehyde for 3-5 min and resuspended in 1 mg / ml X-gal, 40 mM citrate, 40 mM sodium phosphate (pH 6.0), 5 mM potassium iron cyanide β-gal staining solution containing 5 mM ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl ₂ at 37 ° C. for 12-16 hours.

(13) Statistics

Results are expressed as means ± SD. Statistical significance was determined by multiple comparisons at a significance level of P < 0.05 using Student's t-test.

2. Test results

(1) Characterization of immortalized mesenchymal stem cell line

Immortalized mesenchymal stem cell lines (TERT + cells) selected through transduction of hTERT were harvested by transfection and selection using the AMAXA-nucleofactor kit (Lonza Ltd) for electroporation. After immortalization of the native PDSCs, their spindle-shape morphologies were transformed into cuboidal shapes (Fig. 1A). Telomerase activity was not observed in native PDSCs, which was similar to negative control; However, in TRAP analysis, TERT + cells showed strong telomerase activity at the same level as the positive control (Fig. 1B). Nevertheless, RT-PCR analysis did not reveal any difference in the expression of stem cell markers (Figure 1c). In addition, the expression of surface markers of TERT + cells was slightly similar but similar to that of native PDSCs. CDNA, CD105, HLA-ABC, and HLA-DR were negative for hematopoietic markers (e.g., CD34, CD45, and HLA-DR) ) (Fig. 1D and Fig. 1E).

By cell cycle analysis, the proportion of native PDSCs and TERT + cells in the S-phase increased from 17.9% to 41.1%, respectively (Fig. 1f). In addition, the number of TERT + cells after incubation for the same time was significantly higher than the number of native PDSCs (p < 0.005, Fig. 1g). These results indicate that TERT + cells maintain better stem cell characteristics while exhibiting better proliferation compared to natural cells.

In addition, TERT + cells were differentiated into adipocytes by incubation with adipocyte differentiation medium for 21 days, and adipocytes were detected by oil-red O staining (Fig. 2a). Expression of lipoprotein lipase and PPAR-gamma, which are specific markers of adipocytes in differentiated TERT + cells, was also increased compared to native PDSCs (Fig. 2B). In addition, hepatocyte-differentiated TERT + cells were able to detoxify toxins as a representative liver function when exposed to ICG solution (FIG. 2C). By RT-PCR analysis, the expression of hepatocyte-associated genes including albumin and TAT increased after hepatocyte differentiation in native PDSCs; There was no difference in the expression of albumin and TAT between undifferentiated and differentiated cells. AFP expression in TERT + cells decreased after hepatocyte differentiation (Fig. 2D). The efficiency of differentiation of TERT + cells into hepatocytes was lower than that of native PDSCs, but TERT + cells are still expected to have differentiation potential.

(2) Relationship between cell survival and apoptosis in immortalized mesenchymal stem cells

Maintaining a balance between cell survival and apoptosis is an important phenomenon in terms of cell physiology including cell proliferation and differentiation. Thus, through analysis of several markers for stem cell, cell cycle, and death, the role of survival and death in immortalized cells obtained according to the present invention was analyzed. The expression of TERT and stem cell markers including Oct-4 and Nanog was increased in TERT + (Fig. 3a), although the keratoplasm of TERT + cells was modified by insertion of the hTERT gene (Fig. 8). However, there was no difference in the expression of SCF. Compared to native PDSCs, the expression of Cdk-4 and PTTG1 was increased in TERT + cells, and the expression of p53, p21, and cyclin E1 decreased. Interestingly, expression of PTTG1 was significantly increased in TERT + cells, but homologous PTTG2 was not increased (FIG. 3b).

As shown in Figure 3c, the expression of pro-apoptotic genes (e.g., Bax and Caspase-9) was reduced in TERT + cells, except for the cleavage of PARP. Expression of Bcl-2, a typical anti-apoptotic factor, was markedly decreased (FIG. 3c). Also, microtubule-associated protein light chain 3 (LC3), which leads to the formation of a double membrane-limited autophagic vacuole or autophagosome in TERT + cells, , Was reduced through the reduction of phosphorylated mTOR, a negative regulator of type II LC3 (Fig. 3d). These results indicate that immortalized mesenchymal stem cells induce autophosphorylation activity through enhancement of stem cell and cell cycle and reduction of apoptosis pathway including apoptosis and autophagy. FIG. 3E shows that when TERT + is induced in native PDSCs (Naive) cells, the expression of TERT gene is about three times higher than that of Naive cells, whereas the expression of PTTG1 is about four times higher than that of Naive cells.

(3) Effect of down-regulation of PTTG1 by siRNA on autologous proliferation activity of MSCs

As shown above, the expression of PTTG1 was significantly increased in TERT + cells. PTTG1 regulates the cell cycle by preventing separtins from promoting sister chromatid separation in metaphase cell division. In order to evaluate whether proliferative activity enhanced by TERT overexpression correlates with PTTG1 expression, both native PDSCs and TERT + cells were treated with siRNA-PTTG1 and the effect of PTTG1 was analyzed. To confirm downregulation of PTTG1 by siRNA treatment, the expression of PTTG1 was analyzed in both cells. As shown in FIG. 4, the expression of PTTG1 was decreased by siRNA-PTTG1 treatment in both cells, and the expression of PTTG2 was also down-regulated by siRNA-PTTG1 treatment because PTTG2 was homologous to PTTG1 (Fig. 4A). As shown in Fig. 1B, the telomerase activity of TERT + cells was increased as compared to native PDSCs. To confirm the effect of PTTG1 on telomerase activity, telomerase activity was analyzed after siRNA-PTTG1 treatment. Telomerase activity was decreased in TERT + cells after siRNA-PTTG1 treatment compared to non-treated TERT + cells (FIG. 4B, FIG. 4C). In addition, the expression of proliferation-related factors such as Oct-4, HSP90, and Ku70 was reduced in TERT + by downregulation of PTTG1. These results indicate that the expression of PTTG1 is closely related to the telomerase activity as well as the proliferation regulation of TERT + cells. In addition, the expression of Cyclin E1 and p53 as cell cycle regulators was decreased by PTTG1 knockdown, but the expression of Cdk-4 and p21 as cell cycle regulators was increased by down-regulation of PTTG1 in TERT + cells (Fig. 4 ). Regardless of downregulation of PTTG1, there was no difference in the expression of HSP70 in both cells (Fig. 9b).

Next, we analyzed whether PTTG1 expression could inhibit proliferation in native PDSCs and TERT + cells by blocking apoptotic and autophagic pathways. Regardless of the down regulation of PTTG1, there was no difference in expression of Bad and Bax. However, expression of caspase 9 and caspase 3 was increased when PTTG1 was knocked down in both cells. In addition, expression of Bcl-2 was increased in cells treated with siRNA-PTTG1 (FIG. 4E). Analysis of autophagic pathway revealed that expression of phosphorylated mTOR and ATG5-12 was increased in both cells when PTTG1 was down regulated; Type II LC3, an indicator of autoregulatory activity, decreased (Figure 4f). These results suggest that reduced PTTG1 induces apoptosis rather than autopatch.

(4) induction of cell senescence of MSCs by down-regulation of PTTG1

In the serial expansion process, aged MSCs were characterized by a flattened phenotype such as cell senescence and increased? -Galactosidase activity. Down-regulation of PTTG1 induced cell senescence in native PDSCs and TERT + cells (Fig. 5A). Cell cycle analysis was performed after siRNA-PTTG1 treatment in order to determine whether down-regulation of PTTG1 affects cell cycle in both cells. Down-regulation of PTTG1 induced not only senescence but also a decrease in S-phase. In addition, the fraction of sub-G1 TERT + cells was significantly increased (Fig. 5B). In order to confirm the effect of PTTG1 on apoptosis, caspase-3/7 activity was evaluated. There was no difference in caspase-3/7 activity in natural PDSCs due to knockdown of PTTG1. However, caspase-3/7 activity of TERT + cells was increased (FIG. 5C). These results indicate that PTTG1 is involved in cell senescence as well as apoptosis in mesenchymal stem cell lines, indicating that the dual role of PTTG1 may be affected by interaction with TERT.

(5) Functional interaction between TERT and PTTG1

In order to confirm the interaction of PTTG1 and TERT, an immunoprecipitation test was carried out using an anti-TTT antibody. TERT was found to interact with PTTGl as well as HSP90, Ku70, and HSP70 (Figs. 6A, 9C). HSPs have previously been shown to interact with TERT (Haendeler et al. (2003) FEBS Lett 536: 180-186), but the interaction between TERT and PTTG1 has not been elucidated. The present inventors have assumed that the mediator between TERT and PTTG1 affects the dip action of PTTG1 in TERT + cells. Interestingly, it has been reported that Ku heterodimers are associated with telomerase by interactions with hTERT as well as hPTTG (Chai et al. (2002) J Biol Chem 277: 47242-47247). Based on these results, immunoprecipitation using an anti-Ku70 antibody was performed to confirm the interaction between PTTG1 and TERT. As shown in FIG. 6, regardless of PTTG1 expression, HSP90 and Ku70 proteins were found to interact with TERT (FIG. 6A). In addition, Ku70 was found to interact with TERT, PTTG1, and HSP90 (Fig. 6B). Thus, Ku70, TERT, and PTTG1 constitute a complex that affects cell cycle and auto-proliferation. Overexpression of TERT induces the expression of PTTG1, and cell cycle and self regeneration is increased through increased interaction between TERT and PTTG1. However, downregulation of PTTG1 limits its ability to interact with TERT. Therefore, cell death and senescence occurs in PTTG1 down-regulated cells (Figure 7). It is not clear whether this phenomenon is due to the interaction between these two proteins. Based on these results, the present inventors suggest that Ku70 is the mediator between TERT and PTTG1, since Ku70 interacts with both TERT and PTTG1.

3. Discussion

In this study, to overcome the limited lifespan and self regeneration activity of MSCs, an immortalized cell line named TERT + cell was established by inserting and selecting hTERT gene in native PDSCs using AMAXA gene delivery system. We characterized native PDSCs by comparing them to TERT + cells. Characterization of TERT + cells resulted in increased expression of telomerase activity, cell cycle - related factors, and self - extinguishing markers in TERT + cells, but decreased saponin killing. In addition, although TERT + cells showed aneuploid karyotype (Fig. 8), there was no difference in the differentiation potential between native PDSCs and TERT + cells. Interestingly, PTTG1 expression was significantly increased in TERT + cells. By down-regulation of PTTG1, the increase in cell cycle-factor, auto-proliferation, and telomerase activity was reduced; The expression of the apoptotic gene was increased (Fig. 4). In addition, a decrease in PTTG1 induced cell senescence (Fig. 5). From these results, we conclude that PTTG1 is not the only regulatory factor in TERT + cells, and that there may be some regulatory mediator (s) between PTTG1 and TERT.

Cell cycle arrest promotes senescence through cyclin D, cyclin E (Liu et al. (2011) Proc Natl Acad Sci USA 108: 8414-8419), cyclin-dependent kinases CDKs) and upregulation of cyclin D1, cdk 4 and cdk 6 promote the self-renewal of MSCs. In particular, cyclins D1 / E1 increase the self-regulating activity of MSCs through Ca2 + / PKC / MAPKS and PI3K pathways (Ryu et al. (2010) J Cell Physiol 224: 59-70). In this study, it was found that the expression of cyclin E1 is increased by treatment of siRNA-PTTG1 in both native PDSCs and TERT + cells (Fig. 4d).

Pituitary tumor-transforming gene 1 (PTTG1) is a cell cycle-related factor. Up-regulated PTTG1 promotes cell proliferation by increasing the number of cells in the S phase through the MAPK pathway, (2000) Nucleic Acids Res 29: 1300-1307). In addition, it is known that the interaction between DNA and DNA repair is mediated through the interaction between the nucleus and the nucleus. Functionally, PTTG1 modulates telomerase activity through interaction with telomerase, leading to the proper assembly of telomerase enzyme complexes including p23 (Forsythe et al. (2001) J Biol Chem 276 : 15571-15574). In addition, Ku is a heterodimeric protein composed of about 70 kDa subunits and about 80 kDa subunits and is involved in the regulation of telomere maintenance through the telomeric proteins TRF1, TRF2, and TERT binding (Tuteja and Tuteja (2000) Crit Rev Biochem Mol Biol 35: 1-33 O). Chai et al. Also reported that Ku regulate telomerase activity by interaction with TERT (Chai W, et al., (2002) J Biol Chem 277: 47242-47247). However, although the interaction between TERT, HSP70, HSP90, and Ku70 is known, the interaction between TERT and PTTG1 has not been previously characterized. The present inventors demonstrated the interaction between TERT and PTTG1 by immunoprecipitation (FIG. 6). These results indicate that the expression of HSP70 and HSP90 is not altered in response to downregulation of PTTG1 in both cells. These results indicate that PTTG1 does not directly affect the interaction between HSP70, HSP90, and TERT. Nonetheless, the downregulation of PTTG1 reduced Ku70 expression. These results indicate that Ku70 plays an important role as a regulatory mediator between PTTG1 and TERT. TERT interacts with Ku70, and Ku70 also interacts with PTTG1; The resulting complexes translocate to the nucleus, increasing cell cycle, autophagy, and autophosphorylation activity. In contrast, decreased expression of PTTG1 reduces the expression of Ku70 and HSP90. These environmental changes lead to changes in the binding affinity between Ku70 and PTTG1. In addition, the interaction between TERT and Ku70 is reduced by decreased expression of Ku70. These changes lead to cell death and cell senescence as well as aging.

This finding suggests that a balance between TERT and PTTG1 due to efficient coupling with Ku70 may be important for enhancing the limited self-proliferative activity of MSCs, although further study of the regulatory mechanism of Ku70 is needed.

Depositor Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00339

Funding date: 20150324

<110> COLLEGE OF MEDICINE POCHON CHA UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION          GANGNEUNG-WONJU NATIONAL UNIVERSITY INDUSTRY ACADEMY COOPERATION GROUP <120> PROCESS FOR PREPARING IMMORTALIZED MESENCHYMAL STEM CELL LINE <130> PN0728 <160> 34 <170> Kopatentin 2.0 <210> 1 <211> 1093 <212> DNA <213> Homo sapiens <400> 1 cgcgggtggt tagttgagcc ggctccggcg gggaaggagg cgggctgcgg ctgcggctgg 60 ggctgaagct ggggctgggg ttgggggact gcccggggct tagatggctc cgagcccgtt 120 tgagcgtggt ctcggactgc taactggacc aacggcaact gtctgatgag tgccagcccc 180 aaaccgcgcg ctgctcggga ccttagagcc tctgactcag gctggaagat ttgagagctg 240 gattaagtac ttgttggctc acgcccgtga ctgttccgct gtttagctct tgttttttgt 300 gtggacactc ctaggataga aagtttggta tgttgctata cctttgcttc tcccaccttc 360 cccaatatct aatatgtatt tctcattctt agaataatcc agaatggcta ctctgatcta 420 tgttgataag gaaaatggag aaccaggcac ccgtgtggtt gctaaggatg ggctgaagct 480 ggggtctgga ccttcaatca aagccttaga tgggagatct caagtttcaa caccacgttt 540 tggcaaaacg ttcgatgccc caccagcctt acctaaagct actagaaagg ctttgggaac 600 tgtcaacaga gctacagaaa agtctgtaaa gaccaaggga cccctcaaac aaaaacagcc 660 aagcttttct gccaaaaaga tgactgagaa gactgttaaa gcaaaaagct ctgttcctgc 720 ctcagatgat gcctatccag aaatagaaaa attctttccc ttcaatcctc tagactttga 780 gagttttgac ctgcctgaag agcaccagat tgcgcacctc cccttgagtg gagtgcctct 840 catgatcctt gacgaggaga gagagcttga aaagctgttt cagctgggcc ccccttcacc 900 tgtgaagatg ccctctccac catgggaatc caatctgttg cagtctcctt caagcattct 960 gtcgaccctg gatgttgaat tgccacctgt ttgctgtgac atagatattt aaatttctta 1020 gtgcttcaga gtttgtgtgt atttgtatta ataaagcatt ctttaacaga ttcttaaaaa 1080 aaaaaaaaaa aaa 1093 <210> 2 <211> 3829 <212> DNA <213> Homo sapiens <400> 2 caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg gccccggcca cccccgcgat 60 gccgcgcgct ccccgctgcc gagccgtgcg ctccctgctg cgcagccact accgcgaggt 120 gctgccgctg gccacgttcg tgcggcgcct ggggccccag ggctggcggc tggtgcagcg 180 cggggacccg gcggctttcc gcgcgctggt ggcccagtgc ctggtgtgcg tgccctggga 240 cgcacggccg ccccccgccg ccccctcctt ccgccaggtg tcctgcctga aggagctggt 300 ggcccgagtg ctgcagaggc tgtgcgagcg cggcgcgaag aacgtgctgg ccttcggctt 360 cgcgctgctg gacggggccc gcgggggccc ccccgaggcc ttcaccacca gcgtgcgcag 420 ctacctgccc aacacggtga ccgacgcact gcgggggagc ggggcgtggg ggctgctgct 480 gcgccgcgtg ggcgacgacg tgctggttca cctgctggca cgctgcgcgc tctttgtgct 540 ggtggctccc agctgcgcct accaggtgtg cgggccgccg ctgtaccagc tcggcgctgc 600 cactcaggcc cggcccccgc cacacgctag tggaccccga aggcgtctgg gatgcgaacg 660 ggcctggaac catagcgtca gggaggccgg ggtccccctg ggcctgccag ccccgggtgc 720 gaggaggcgc gggggcagtg ccagccgaag tctgccgttg cccaagaggc ccaggcgtgg 780 cgctgcccct gagccggagc ggacgcccgt tgggcagggg tcctgggccc acccgggcag 840 gcgcgtgga ccgagtgacc gtggtttctg tgtggtgtca cctgccagac ccgccgaaga 900 agccacctct ttggagggtg cgctctctgg cacgcgccac tcccacccat ccgtgggccg 960 ccagcaccac gcgggccccc catccacatc gcggccacca cgtccctggg acacgccttg 1020 tcccccggtg tacgccgaga ccaagcactt cctctactcc tcaggcgaca aggagcagct 1080 gcggccctcc ttcctactca gctctctgag gcccagcctg actggcgctc ggaggctcgt 1140 ggagaccatc tttctgggtt ccaggccctg gatgccaggg actccccgca ggttgccccg 1200 cctgccccag cgctactggc aaatgcggcc cctgtttctg gagctgcttg ggaaccacgc 1260 gcagtgcccc tacggggtgc tcctcaagac gcactgcccg ctgcgagctg cggtcacccc 1320 agcagccggt gtctgtgccc gggagaagcc ccagggctct gtggcggccc ccgaggagga 1380 ggacacagac ccccgtcgcc tggtgcagct gctccgccag cacagcagcc cctggcaggt 1440 gtacggcttc gtgcgggcct gcctgcgccg gctggtgccc ccaggcctct ggggctccag 1500 gcacaacgaa cgccgcttcc tcaggaacac caagaagttc atctccctgg ggaagcatgc 1560 caagctctcg ctgcaggagc tgacgtggaa gatgagcgtg cgggactgcg cttggctgcg 1620 caggagccca ggggttggct gtgttccggc cgcagagcac cgtctgcgtg aggagatcct 1680 ggccaagttc ctgcactggc tgatgagtgt gtacgtcgtc gagctgctca ggtctttctt 1740 ttatgtcacg gagaccacgt ttcaaaagaa caggctcttt ttctaccgga agagtgtctg 1800 gagcaagttg caaagcattg gaatcagaca gcacttgaag agggtgcagc tgcgggagct 1860 gtcggaagca gaggtcaggc agcatcggga agccaggccc gccctgctga cgtccagact 1920 ccgcttcatc cccaagcctg acgggctgcg gccgattgtg aacatggact acgtcgtggg 1980 agccagaacg ttccgcagag aaaagagggc cgagcgtctc acctcgaggg tgaaggcact 2040 gttcagcgtg ctcaactacg agcgggcgcg gcgccccggc ctcctgggcg cctctgtgct 2100 gggcctggac gatatccaca gggcctggcg caccttcgtg ctgcgtgtgc gggcccagga 2160 cccgccgcct gagctgtact ttgtcaaggt ggatgtgacg ggcgcgtacg acaccatccc 2220 ccaggacagg ctcacggagg tcatcgccag catcatcaaa ccccagaaca cgtactgcgt 2280 gcgtcggtat gccgtggtcc agaaggccgc ccatgggcac gtccgcaagg ccttcaagag 2340 ccacgtctct accttgacag acctccagcc gtacatgcga cagttcgtgg ctcacctgca 2400 ggagaccagc ccgctgaggg atgccgtcgt catcgagcag agctcctccc tgaatgaggc 2460 cagcagtggc ctcttcgacg tcttcctacg cttcatgtgc caccacgccg tgcgcatcag 2520 gggcaagtcc tacgtccagt gccaggggat cccgcagggc tccatcctct ccacgctgct 2580 ctgcagcctg tgctacggcg acatggagaa caagctgttt gcggggattc ggcgggacgg 2640 gctgctcctg cgtttggtgg atgatttctt gttggtgaca cctcacctca cccacgcgaa 2700 aaccttcctc agctatgccc ggacctccat cagagccagt ctcaccttca accgcggctt 2760 caaggctggg aggaacatgc gtcgcaaact ctttggggtc ttgcggctga agtgtcacag 2820 cctgtttctg gatttgcagg tgaacagcct ccagacggtg tgcaccaaca tctacaagat 2880 cctcctgctg caggcgtaca ggtttcacgc atgtgtgctg cagctcccat ttcatcagca 2940 agtttggaag aaccccacat ttttcctgcg cgtcatctct gacacggcct ccctctgcta 3000 ctccatcctg aaagccaaga acgcagggat gtcgctgggg gccaagggcg ccgccggccc 3060 tctgccctcc gaggccgtgc agtggctgtg ccaccaagca ttcctgctca agctgactcg 3120 acaccgtgtc acctacgtgc cactcctggg gtcactcagg acagcccaga cgcagctgag 3180 tcggaagctc ccggggacga cgctgactgc cctggaggcc gcagccaacc cggcactgcc 3240 ctcagacttc aagaccatcc tggactgatg gccacccgcc cacagccagg ccgagagcag 3300 acaccagcag ccctgtcacg ccgggctcta cgtcccaggg agggaggggc ggcccacacc 3360 caggcccgca ccgctgggag tctgaggcct gagtgagtgt ttggccgagg cctgcatgtc 3420 cggctgaagg ctgagtgtcc ggctgaggcc tgagcgagtg tccagccaag ggctgagtgt 3480 ccagcacacc tgccgtcttc acttccccac aggctggcgc tcggctccac cccagggcca 3540 gcttttcctc accaggagcc cggcttccac tccccacata ggaatagtcc atccccagat 3600 tcgccattgt tcacccctcg ccctgccctc ctttgccttc cacccccacc atccaggtgg 3660 agaccctgag aaggaccctg ggagctctgg gaatttggag tgaccaaagg tgtgccctgt 3720 acacaggcga ggaccctgca cctggatggg ggtccctgtg ggtcaaattg gggggaggtg 3780 ctgtgggagt aaaatactga atatatgagt ttttcagttt tgaaaaaaa 3829 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 agtgagaggc aacctggaga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gtgaagtgag ggctcccata 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 ttcttgactg ggaccttgtc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gcttgccttg ctttgaagca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 agaaccccaa gatgcacaac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gggcagcgtg tacttatcct 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 gagtgaaatg gcacgatacc ta 22 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 tttcctctcc ttcttcttca ccttc 25 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 ggagttatgg tgggtatggg tc 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 agtggtgaca aaggagtagc ca 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 atgctgcaaa ctgaccacgc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 gcttcgcttt gccaatgctt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 gcggctacta caaccagagc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 gcacatggca cgtgtatctc 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 gtccgtggct acctgtcat 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 18 agccctttct caaaggcttc 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 ggaaagacaa caaacaaatc ac 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 20 tgcattgaac ttcacagcaa ac 22 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 atgctgcaaa ctgaccacgc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 gcttcgcttt gccaatgctt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 23 ccccaagtgt caactccaac 20 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 24 ccagaagaca ccctccaaag gac 23 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 25 aacgatgtgg agttcacgg 19 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 26 gacacatcct caggagaatg g 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 27 aaggaaaatg gagaacaggc 20 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 28 gcttggctgt ttttgtttga gg 22 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 29 ctgatctacg ttgataagga a 21 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 30 ctatttctgg ataggcgtca tctg 24 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 31 tccttctgca tcctgtcagc a 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 32 caggagatgg ccactgccgc a 21 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 33 aatccgtcga gcagagtt 18 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 34 cccttaccct tacccttacc ctaa 24

Claims (9)

(a) providing mesenchymal stem cells derived from the chorionic plate of the placenta; And (b) separating cells expressing the PTTG1 gene by separating cuboidal cells from cells obtained by transfecting the mesenchymal stem cells derived from the placenta placenta plate with the gene of SEQ ID NO: 2 &Lt; / RTI &gt; wherein the transformed mesenchymal stem cell line is transformed into a transformed mesenchymal stem cell line. delete delete delete delete The method according to claim 1, wherein said transfection is carried out by electroporation. delete delete Immortalized placental chorionic plate-derived mesenchymal stem cell line KCLRF-BP-00339.

Images