KR101798217B1 - Sorting method of mesenchymal stem cell derived from pluripotent stem cell - Google Patents

Abstract

Provided is a method for effectively sorting mesenchymal stem cells derived from pluripotent stem cells. The method comprises the following steps: spontaneously differentiating pluripotent stem cells into differentiated cells including ectoderm cells, mesoblast cells and endodermal cells; cultivating the differentiated cells in a fibronectin cultivation container using a mesenchymal stem cell cultivation medium; and separating the mesenchymal stem cells attached on the fibronectin cultivation container in a natural selection manner.

Description

TECHNICAL FIELD The present invention relates to a method for screening mesenchymal stem cells derived from pluripotent mesenchymal stem cells (SORTING METHOD OF MESENCHYMAL STEM CELL DERIVED FROM PLURIPOTENT STEM CELL)

The present invention relates to a method for efficiently screening mesenchymal stem cells derived from pluripotent stem cells.

Various methods for separating adult mesenchymal stem cell (MSC) from various tissues such as bone marrow, peripheral blood, fat, umbilical cord blood, skin tissue, intervertebral disc, amniotic fluid, placenta and periodontal tissue have been developed, Cells have been used for therapy by inducing culture and proliferation of isolated cells for use as regenerative medicine and cell therapy agents.

In order to use mesenchymal stem cells as regenerative medicine and cell therapy agents, a large number of high-quality and high-quality cells are required to meet the respective treatment conditions and the minimum number of cells required for treatment (about 1x10 ⁹ cells) need. However, the adult mesenchymal stem cells obtained from the adult are different in their differentiation ability depending on the tissue and donor, and also have a limit of low cell proliferation ability.

The most ideal alternative for resolving the limitations of cells due to the differentiation ability and the cell proliferation capacity of existing adult mesenchymal stem cells is to use a pluripotent mesenchymal stem cell Cells.

However, the technology to date has limitations in effectively classifying mesenchymal stem cells derived from pluripotent stem cells, and various techniques are required to solve the problems.

A method for efficiently screening mesenchymal stem cells derived from pluripotent stem cells is provided.

Methods for efficiently selecting mesenchymal stem cells derived from pluripotent stem cells include spontaneous differentiation of pluripotent stem cells into differentiated cells, culturing the differentiated cells in a fibronectin- fibronectin_coated culture container, and naturally selectively separating the mesenchymal stem cells attached to the fibronectin_culturing vessel.

The pluripotent stem cells can be cultured in a feeder cell-free or feeder cell environment. The pluripotent stem cells may be at least one of a human embryonic stem cell and a human induced pluripotent stem cell.

The differentiated cells can be cultured in the fibronectin-culture vessel using mesenchymal stem cell culture medium. The differentiated cells include ectodermal cells, mesodermal cells and endoderm cells.

The details of other embodiments are included in the detailed description and drawings.

The invention has at least the following effects.

Mesenchymal stem cells derived from pluripotent stem cells can be efficiently screened.

An efficient selection of mesenchymal stem cells derived from pluripotent stem cells can provide a platform in which a sufficient number of high quality cell lines necessary for regenerative therapy can be easily obtained.

The effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the specification.

FIG. 1 is a graph showing the results of cultured mesenchymal stem cells (control cells: Control) cultured in a general culture container (hereinafter referred to as 'general culture container') not coated with the extracellular matrix, (Gelatin, PLL, Fn) of the mesenchymal stem cells (experimental cells) cultured in a culture medium (hereinafter referred to as " coating culture container "
FIG. 2 is an image showing differences in cell adhesion efficiency between control cells cultured in a general culture vessel and experimental cells (Gelatin, PLL, Fn) cultured in a coating culture vessel.
FIG. 3 is a graph showing the effect of a control cell (control) cultured in a general culture vessel and an experimental cell (Gelatin, PLL, Fn) cultured in a coating culture vessel using a fluorescence activated cell sorter (FACS) This is an image of the result of confirming the expression of human mesenchymal stem cell surface markers CD90 and CD105.
FIG. 4 shows immunocytochemistry (ICC) results of control cells cultured in a general culture vessel and experimental cells (Gelatin, PLL, Fn) cultured in a coating culture vessel using a confocal microscope Image.
FIG. 5 is a graph showing the relationship between control cells (control) cultured in a general culture vessel and experimental cells (Gelatin, PLL (control)) cultured in a coating culture vessel using a real-time polymerase chain reaction , Fn), and the results of confirming the expression of integrin alpha 1, integrin alpha 4, integrin alpha 5, integrin alpha V and integrin beta 1.
FIG. 6 is a graph showing the results of a cell adhesion test using an integrin antibody in differentiated cells. In the control group, no integrin antibody was used and in the experimental group (? 5,? 1,? 5? 1), integrin? 5 antibody, The integrin [alpha] 5 [beta] l antibody was used.
FIG. 7 is a quantitative analysis graph of the cell adhesion ability test results of FIG. 6. FIG.
FIG. 8 is a graph showing the results of the detection of mesenchymal stem cell surface markers expressed on selected Fn-separated MSCs using a fluorescence activated cell sorter (FACS) using a fibronectin Adipose-derived MSCs (ASC) and bone marrow-derived mesenchymal stem cells (Bone Marrow MSC, BM-MSC) were used as controls.
FIG. 9 is a graph showing the results of differentiation of mesenchymal stem cells (ES cell-derived cells: ES-Fn-MSC) into adipocytes by using a fibronectin- Bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.
Fig. 10 shows the results of the analysis of lipid differentiation markers (PPARγ, C / EBPβ) expressed in selected mesenchymal stem cells (ES-Fn-MSC) using real-time PCR using a fibronectin As an image of the results of analysis, fat-derived mesenchymal stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.
FIG. 11 is an image showing the results of differentiation of mesenchymal stem cells (ES cell-derived cells: ES-Fn-MSC) into osteocytes selected using a fibronectin- And bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.
Figure 12 shows the results of analysis of bone morphogenetic markers (Col1, ALP) expressed in selected mesenchymal stem cells (ES-Fn-MSC) using real-time PCR using Fibronectin As an image of the results, fat-derived mesenchymal stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.
FIG. 13 is an image of the result of differentiation of mesenchymal stem cells (ES-Fn-MSC) selected into chondrocytes by using a fibronectin-culture vessel. As shown in FIG. 13, fat-derived mesenchymal stem cells (ASC) And bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.
Fig. 14 shows the results of analysis of cartilage differentiation markers (Col2, AGG) expressed in selected mesenchymal stem cells (ES-Fn-MSC) using real-time PCR using a fibronectin As an image of the results, fat-derived mesenchymal stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.
FIG. 15 is an image showing the results of live / dead assay of mesenchymal stem cells (ES cell-derived cells: ES-Fn-MSC) selected using a fibronectin-culture container.
16 is a graph showing the results of confirming the cell proliferation ability of mesenchymal stem cells (experimental group cells: ES-Fn-MSC) cultured in a fibronectin_-culture container through a hematocytometer, wherein the fat- Leaf stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.
17 is an image showing the results of analysis of the cell cycle of mesenchymal stem cells (experimental cell: ES-Fn-MSC (p3)) cultured in a fibronectin_-culture container using a fluorescence activated cell sorter (FACS) Derived mesenchymal stem cells (ASC (p3)) and bone marrow-derived mesenchymal stem cells (BM-MSC (p3)) were used as controls.
18 is a graph showing the results of measurement of telomere length of mesenchymal stem cells (experimental cell: ES-Fn-MSC) cultured in a fibronectin_-culture vessel using real-time PCR, Leaf stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.

BRIEF DESCRIPTION OF THE DRAWINGS The features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

It will be apparent to those skilled in the art that the present invention is not limited to the embodiments disclosed herein, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, And the scope of the invention is to be defined only by the scope of the claims.

Further, in the description of the technology disclosed in this specification, a detailed description of related art may be omitted if it is judged that the gist of the technology disclosed in this specification may be blurred.

Throughout this specification, when an element is referred to as being " comprising " or "having" an element, it is understood that it does not exclude other elements, it means.

Throughout this specification, "pluripotent stem cells" are cells that are somewhat more advanced than embryonic cells, for example, the inner cell mass of the blastocyst (ICM) . The pluripotent stem cell refers to an undifferentiated cell capable of differentiating into an endoderm cell, a mesoderm cell, and an ectodermal cell, and is understood to include both embryonic stem cells and inducible pluripotent stem cells.

Throughout this specification, "human embryonic stem cell (hESC)" is a cell culture obtained by extracting the inner cell mass from a blastocyst embryo immediately before fertilization of the embryo into the uterus of the mother, Which is capable of pluripotency.

Throughout this specification, the term " human induced pluripotent stem cell (hiPS) "refers to a cell induced to have pluripotency, such as human embryonic stem cells, by introducing a gene that induces de- differentiation into fully- do.

Throughout this specification, "differentiation" means that the cell develops to the level of a specific cell or tissue complex or entity having a particular function.

Throughout this specification, "differentiated cells" means endoderm cells, mesoderm cells, cells that have spontaneously differentiated into ectodermal cells.

Throughout this specification, "spontaneous differentiation" refers to a process that is naturally occurring without treating growth factors involved in cell differentiation or treating certain chemicals, treating growth factors involved in cell differentiation or treating certain chemicals And is different from guided differentiation that induces differentiation of embryonic stem cells into desired cells.

Throughout this specification, "feeder cell-free culture" refers to the use of an extracellular matrix (ECM), a recombinant protein, or a synthetic biomaterial to produce mouse embryonic fibroblasts mouse embryonic fibroblast) to replace human embryonic stem cells.

Throughout this specification, "A_ culture vessel" means a culture vessel coated with A. For example, "fibronectin_culture vessel" means a culture vessel coated with fibronectin, and "gelatin_culture vessel" means a culture vessel coated with gelatin.

Throughout this specification, "mesenchymal stem cell" is a multipotent stem cell having the characteristics of mesenchymal stem cells and capable of differentiating into adipocytes, chondrocytes, bone cells, etc., As well as pseudocytes with properties similar to those of mesenchymal stem cells. Thus, "mesenchymal stem cells" can be used to refer to mesenchymal stem cells themselves, mesenchymal stem cell-like cells themselves having similar characteristics to mesenchymal stem cells, or mixtures thereof, respectively. The mesenchymal stem cell-like cell may be at least one of the cells having the characteristics of mesenchymal stem cells, for example, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more.

Throughout this specification, "natural selective isolation" means that the mesenchymal stem cells are naturally attached to the fibronectin.

Hereinafter, the present invention will be described in more detail with reference to the embodiments described below and the accompanying drawings.

The present invention relates to a method of spontaneous differentiation of pluripotent stem cells into differentiated cells, culturing the differentiated cells in a fibronectin_coated culture container using mesenchymal stem cell culture medium And naturally selectively classifying the mesenchymal stem cells attached to the fibronectin-culture vessel.

The pluripotent stem cells may be at least one of human embryonic stem cells and human induced pluripotent stem cells, and the differentiated cells include ectodermal cells, mesodermal cells and endoderm cells.

The inventors have experimentally confirmed that the mesenchymal stem cells derived from the pluripotent stem cells can be efficiently classified using the fibronectin-culture container, and the mesenchymal stem cells attached to the fibronectin- , Mesenchymal stem cells, and mesenchymal stem cells adhered to poly-L-lysine-cultured cells, and the cell proliferation capacity was higher than that of mesenchymal stem cells. This will be described in detail with reference to Figs. 1 to 7.

First, referring to FIG. 1 and FIG. 2, the cell adhesion ability of mesenchymal stem cells to different extracellular matrix will be described.

FIG. 1 is a graph showing the relationship between the number of mesenchymal stem cell-like cells (control cells: control) cultured in a general culture container (hereinafter referred to as a 'general culture container') not coated with the extracellular matrix and the culture container (Gelatin, PLL, Fn) of mesenchymal stem cell-like cells cultured in a 'coating culture container'.

(PLL) refers to mesenchymal stem cells cultured in a poly-L-lysine culture dish, and an experimental group cell (Fn ) Refers to mesenchymal stem cells cultured in a fibronectin-culture vessel.

FIG. 2 is an image showing differences in cell adhesion efficiency between control cells cultured in a general culture vessel and experimental cells (Gelatin, PLL, Fn) cultured in a coating culture vessel.

Referring to FIGS. 1 and 2, cell adhesion to the extracellular matrix was significantly higher in the experimental group (Fn) than in the other experimental cells (Gelatin, PLL) and control cells (Control). Specifically, the test cell (Fn) showed about 3 times more cell adhesion ratio than the other test cell (PLL).

Referring to FIG. 3, it will be explained that the test cell (Fn) shows stronger characteristics of mesenchymal stem cells compared to other experimental cells (Gelatin, PLL) and control cells (Control).

FIG. 3 is a graph showing the effect of a control cell (control) cultured in a general culture vessel and an experimental cell (Gelatin, PLL, Fn) cultured in a coating culture vessel using a fluorescence activated cell sorter (FACS) CD90 < / RTI > and < RTI ID = 0.0 > CD105. ≪ / RTI >

3, the surface markers (CD90, CD105) of the mesenchymal stem cells were all positive in the test cell (Fn), and the CD105 was more than 64%. Table 1 summarizes the expression rates of surface markers (CD90, CD105) of mesenchymal stem cells expressed in control cells and control cells (Gelatin, PLL, Fn).

Control Gelatin PLL Fn CD90 61.9% 76.5% 76.2% 75.5% CD105 25.2% 30.6% 42.7% 64.4%

From the results shown in the above Table 1, the experimental cell (Fn) and the experimental cell (Gelatin) can satisfy the following formula 1, and the experimental cell (Fn) and the experimental cell (PLL)

<Formula 1>

<Formula 2>

In the above Equations 1 and 2, CD105 (Fn) is the CD105 expression level of mesenchymal stem cells adhered to the fibronectin_culture container, CD105 (G) is the CD105 expression level of the mesenchymal stem cells adhered to the gelatin_culture container , And CD105 (PLL) is the expression rate of CD105 in mesenchymal stem cells attached to a poly-L-lysine culture dish.

FIG. 4 shows immunocytochemistry (ICC) results of control cells cultured in a general culture vessel and experimental cells (Gelatin, PLL, Fn) cultured in a coating culture vessel using a confocal microscope Image.

4, the staining of the locally attached protein vinculin and the cytoskeletal protein actin was significantly lower than that of control cells (Gelatin, PLL) compared to control cells (Fn ). These results indicate that the test cell (Fn) is superior to other experimental cells (Gelatin, PLL) in cell proliferation and cell regeneration ability.

Vinculin is a protein that binds the cytoskeleton and extracellular matrix (ECM), and is one of the regulators of cartilage differentiation. When vinculin function is inhibited, Col2 expression is reduced in chondrocytes, and p38 MAPK inhibition (Koshimizu T et al., Vinculin functions as regulator of chondrogenesis. J BioChem 2012; 287: 15760-75).

In addition, vinculin plays an important role in regenerating myotubule for the regeneration of myocytes, and hyperchulinemia-free embryonic stem cells are essential for cell attachment and cell function It is known that no changes are observed, the cells do not migrate to where they are needed, and the genes necessary for differentiation are not expressed (Mierke, CT (2009). Cell Biochem Biophys 53, 115-126).

On the other hand, the inventors confirmed that the test cell (Fn) was adherent to the fibronectin-culture vessel via the integrin [alpha] 5 [beta] 1. Hereinafter, a detailed description will be given with reference to Figs. 5 to 7. Fig.

FIG. 5 is a graph showing the relationship between control cells (control) cultured in a general culture vessel and experimental cells (Gelatin, PLL (control)) cultured in a coating culture vessel using a real-time polymerase chain reaction , Fn) of integrin? 1, integrin? 4, integrin? 5, integrin? V and integrin?

FIG. 6 is a graph showing the results of the cell adhesion test using an integrin antibody. In the control group, no integrin antibody was used. In the experimental group (? 5,? 1,? 5? 1), integrin? 5 antibody, integrin? 1 antibody and integrin? Was used.

The experimental group (α5) inhibited the adhesion of mesenchymal stem cells to the extracellular matrix using the integrin α5 antibody. In the experimental group (β1), the adhesion of the mesenchymal stem cells to the extracellular matrix using the integrin β1 antibody (Α5β1) inhibited cell adhesion of mesenchymal stem cells to extracellular matrix using integrin α5β1 antibody.

FIG. 7 is a quantitative analysis graph of the cell adhesion ability test results of FIG. 6. FIG.

5 to 7, except for integrin? 4, integrin? 1, integrin? 5 and integrin? V were most expressed in experimental cells (Fn), and in particular, in integrin cells (Fn) . On the other hand, as a result of testing the cell adhesion ability using the integrin antibody, the cell adhesion rate of mesenchymal stem cells was lower in the experimental group (? 5? 1) than the experimental group (? 5). It was confirmed that the mesenchymal stem cells adhering to the experimental group (Fn), that is, the fibronectin _ culture container, were naturally selectively classified via the integrin α5β1.

On the other hand, the inventors confirmed that the mesenchymal stem cells selected through the fibronectin-culture container were attached to the fibronectin-culture vessel in the form of fibroblasts characteristic of mesenchymal stem cells.

In addition, the inventors of the present invention have found that, in mesenchymal stem cells cultured in a fibronectin-culture container, the profile of surface markers of mesenchymal stem cells is similar to that of fat-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells Respectively. Hereinafter, this will be described in detail with reference to FIG.

FIG. 8 is a graph showing the results of screening of mesenchymal stem cell surface markers expressed in Fn-separated MSCs selected through a fluorescence-activated cell sorter (FACS) Adipose-derived MSCs (ASCs) and bone marrow-derived mesenchymal stem cells (Bone Marrow MSCs, BM-MSCs) were used as controls.

8, the positive markers of mesenchymal stem cells, CD44, CD90, and CD105 showed a high expression rate of 95% or higher, and OCT4, an undifferentiated marker of embryonic stem cells, and CD31, CD34, The expression levels of OCT4, CD31, CD34, HLA-DR, CD44, CD90 and CD105 were significantly lower than those of Fn-separated MSC and control cells (ASC, BM- MSC).

The inventors have found that mesenchymal stem cells cultured in a fibronectin-cultured vessel have high differentiation ability compared to fat-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells, and are capable of differentiating into adipocytes, bone cells, cartilage cells and the like And it can be differentiated into.

Hereinafter, this will be described in detail with reference to FIG. 9 to FIG.

FIG. 9 is an image of the result of differentiation of mesenchymal stem cell-like cells (experimental group cell: ES-Fn-MSC) cultured in a fibronectin_culture container into adipocytes, wherein fat-derived mesenchymal stem cells (ASC) And bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.

10 shows the results of analysis of lipid differentiation markers (PPARγ, C / EBPβ) expressed in mesenchymal stem cells (ES-Fn-MSC) cultured in a fibronectin_ culture container using real-time PCR As an image of the results, fat-derived mesenchymal stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.

9 and 10, the adipocyte differentiation markers (PPARγ and C / EBPβ) were observed in the test cell group (ES-Fn-MSC) as compared to the fat-derived mesenchymal stem cell (ASC) And was also highly expressed.

FIG. 11 is an image of the result of differentiation of mesenchymal stem cells (experimental group cells: ES-Fn-MSC) into osteocytes cultured in a fibronectin_multi culture vessel, wherein fat-derived mesenchymal stem cells (ASC) - derived mesenchymal stem cells (BM-MSC) were used as controls.

12 shows the results of analysis of bone morphogenetic markers (Col1, ALP) expressed in mesenchymal stem cells (experimental cell: ES-Fn-MSC) cultured in a fibronectin_2 culture container using real-time PCR As an image, fat-derived mesenchymal stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.

11 and 12, osteoclast differentiation ability was confirmed in mesenchymal stem cells (ES-Fn-MSC) as compared with bone marrow-derived mesenchymal stem cells (BM-MSC) ALP) was also highly expressed.

FIG. 13 is an image of the result of differentiation of mesenchymal stem cells (experimental group cells: ES-Fn-MSC) into chondrocytes cultured in a fibronectin_multi culture vessel, wherein fat-derived mesenchymal stem cells (ASC) - derived mesenchymal stem cells (BM-MSC) were used as controls.

14 shows the results of analysis of cartilage differentiation markers (Col2, AGG) expressed in mesenchymal stem cells (experimental cell: ES-Fn-MSC) cultured in a fibronectin_ culture container using real-time PCR As an image, fat-derived mesenchymal stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.

13 and 14, high chondrocyte cell differentiation ability was confirmed in mesenchymal stem cells (ES-Fn-MSC) as compared with bone marrow-derived mesenchymal stem cells (BM-MSC) And AGG) were also highly expressed.

Further, the inventors confirmed that mesenchymal stem cells selected through the fibronectin_multi-culture vessel have higher cell proliferation capacity and longer cell life than the fat-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells Respectively. This will be described with reference to FIGS. 15 to 18. FIG.

15 is an image showing the result of a live / dead assay of mesenchymal stem cells (test cell: ES-Fn-MSC) cultured in a fibronectin-culture container.

16 is a graph showing the results of confirming the cell proliferation ability of mesenchymal stem cells (experimental group cells: ES-Fn-MSC) cultured in a fibronectin_-culture container through a hematocytometer, wherein the fat- Leaf stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.

17 is an image showing the results of analysis of the cell cycle of mesenchymal stem cells (experimental cell: ES-Fn-MSC (p3)) cultured in a fibronectin_-culture container using a fluorescence activated cell sorter (FACS) Derived mesenchymal stem cells (ASC (p3)) and bone marrow-derived mesenchymal stem cells (BM-MSC (p3)) were used as controls.

15 and 16, the cell survival rate of ES-Fn-MSC was high and the cell proliferation rate was higher than that of control cells (ASC, BM-MSC) as the number of passages increased. In addition, as shown in Fig. 17, the cells of the S and G2 / M group, which are associated with cell division, are observed in the third passage (ES-Fn-MSC Cells (ASC (p3), BM-MSC (p3)), respectively.

18 is a graph showing the results of measurement of telomere length of mesenchymal stem cells (experimental cell: ES-Fn-MSC) cultured in a fibronectin_-culture vessel using real-time PCR, Leaf stem cells (ASC) and bone marrow-derived mesenchymal stem cells (BM-MSC) were used as controls.

Referring to FIG. 18, it was found that the length of the telomere in the test cell (ES-Fn-MSC) was longer than that of the control cell (ASC, BM-MSC). From the results shown in Fig. 18, it can be seen that the test cell (ES-Fn-MSC) satisfies the following equations 3 and 4.

<Formula 3>

T / S (ASC) < T / S (ES-Fn-MSC)

<Formula 4>

T / S (BM-MSC) < T / S (ES-Fn-MSC)

(BM-MSC) is the telomere length of the bone marrow-derived mesenchymal stem cells, T / S (ASC) is the telomere length of the fat-derived mesenchymal stem cells, (ES-Fn-MSC) is the telomere length of mesenchymal stem cells.

The pluripotent stem cells can be cultured in a supportive cell or feeder cell-free environment. The above-mentioned non-supporting cell culture can be used for culturing embryonic stem cells using conventional supporting cells such as mouse embryonic fibroblasts, for example, in the screening of new drugs or in the evaluation of efficacy and toxicity, And the occurrence of errors in the analysis of the experimental results can be improved. For example, the non-supporting cell environment can be Matrigel ( ^TM ) as an extracellular matrix, and E8 medium or mTeSR1 maintenance medium ^TM can be used as a medium.

The medium used for the differentiation of the pluripotent stem cells and the culture of the mesenchymal stem cells may be a general medium used for culturing the stem cells, preferably a serum (for example, fetal bovine serum, horse serum and human (Eagles ' s minimum essential medium, Eagle, H. Science 130: 432 (1959)), alpha-MEM (Stanner, CP et al. , 1976), 199 medium (Morgan et al., Proc. Soc. (1986)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 1963), F12 (Ham, Proc. Natl., &Lt; RTI ID = 0.0 &gt; Dulbecco's Modification of Eagle's Medium, Dulbecco, R. et al., Vir. Proc. USA 53: 288 (1965)), F10 (Ham, RG Exp. Cell Res. 29: 515 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mo, h's MB752 / 1 (Waymo, h, C 100: 115 (1959)) and the MCDB series (Ham et al., Proc. Soc. Exp. Biol. RG et al., In Vitro 14: 11 (1978)). The medium may also include components such as, for example, antibiotics or antifungal agents (e.g., penicillin, streptomycin), nitrogen sources and glutamine.

For example, the differentiation medium used for the differentiation of the pluripotent stem cells is DMEM / F12 containing 10% fetal bovine serum, 1% non essential amino acid (NEAA) and 1% penicillin / strapthomycin (P / S) (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12).

For example, the mesenchymal stem cell culture medium may be Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (P / S) have.

Hereinafter, embodiments and experimental examples will be described in detail.

&Lt; Example 1 >

(1) Culture of human embryonic stem cells

(MatrigelTM, BD Bioscience, Cat. 356234) was diluted 100-fold with serum-free culture medium (Serum free DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12)). 3 mL of Matrigel solution diluted 100 times with DMEM / F12 was added to a 60 mm culture container and coating was carried out at 4 캜 for 12 hours or more. After coating was completed, the 60 mm Matrigel coating incubation vessel was washed once with DPBS, and E8 (Essential 8) medium was added thereto. Thereafter, the 60 mm Matrigel coating incubation vessel was stored in an incubator.

The following two methods were used to transfer undifferentiated human embryonic stem cells (H9 hESC) to a 60 mm Matrigel-coated culture vessel. First, human embryonic stem cells (H9 hESC) in undifferentiated state are divided into about 100 small lumps using a micropipette, and then transferred to a 60 mm Matrigel coating incubator. As another method, human embryonic stem cells (H9 hESC) in undifferentiated state were made into single cell level using EDTA, and then cultured in a 60 mm Matrigel coating incubation vessel as a ROCK inhibitor with Y-27632 (Y-27632 dihydrochloride, CAS No. 129830-38-2) is added to 1 μL per 3 mL of E8 medium.

Then, a 60 mm Matrigel coating incubation vessel was placed in an incubator at 37 ° C in a 5% CO ₂ environment, and E8 medium was added once a day until human embryonic stem cells were grown to 70% (about 3 to 4 days) And human embryonic stem cells were cultured.

(2) Differentiation of human embryonic stem cells

After human embryonic stem cells were grown to 70%, the 60 mm Matrigel coating incubation vessel was treated with differentiation medium for 7 days at 37 ° C, 5% CO ₂ environment. The differentiation medium was replaced daily for 7 days. The differentiation medium was DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) containing 10% fetal bovine serum, 1% non essential amino acid (NEAA) and 1% penicillin / Respectively.

(3) Adhesion induction and maintenance culture of mesenchymal stem cells

On the eighth day of differentiation, the differentiated cells were washed once with DPBS, and then made into single cells using trypsin. Then, the lump cells were removed using a 40 μm cell strainer, the remaining differentiated cells were centrifuged at 1200 rpm for 3 minutes, and then the supernatant was removed. After removing the supernatant, MSC medium was added to the remaining differentiated cells to harvest residual differentiated cells. Then, the harvested residual differentiated cells were transferred to a fibronectin-culture vessel, and the fibronectin-culture vessel was incubated at 37 DEG C in a 5% CO ₂ environment for 12 hours.

After 12 hours of culture, the supernatant was removed, and a new mesenchymal stem cell culture medium was added thereto. The mesenchymal stem cells were cultured for 5 days in a 5% CO ₂ environment at 37 ° C.

For mesenchymal stem cell culture medium, DMEM-H (Dulbecco's Modified Eagle's Medium with high glucose) containing 10% fetal bovine serum and 1% penicillin / streptomycin (P / S) was used.

&Lt; Example 2 >

Mesenchymal stem cells were induced and cultured in the same manner as in (1) to (3) of Example 1, except that the residual differentiated cells harvested in Example 1 were transferred to a gelatin-culture vessel.

&Lt; Example 3 >

(1) to (3) of Example 1, except that the residual differentiated cells harvested in Example 1 were transferred to a poly-L-lysine culture dish. Lt; / RTI &gt;

&Lt; Comparative Example 1 &

(1) to (3) of Example 1, except that the residual differentiated cells harvested in Example 1 were transferred to a general culture vessel not coated with the extracellular matrix, And cultured.

< Experimental Example  1. Different The extracellular matrix  Cultured in an applied culture container Of mesenchymal stem cells  Characteristic evaluation>

The characteristics of the experimental group (Fn) obtained in Example 1, the experimental group (Gelatin) obtained in Example 2 and the experimental group (PLL) obtained in Example 3 were evaluated.

Experimental cells (Fn) were prepared from mesenchymal stem cells (H9 hESc) and cultured in a medium of 5% CO ₂ at 37 ° C for 12 hours using a mesenchymal stem cell culture medium, Lt; / RTI &gt;

Experimental group (Gelatin) was mesenchymal stem cells isolated from human embryonic stem cells (H9 hESc) and cultured in a gelatin-culture vessel using mesenchymal stem cell culture medium for 12 hours at 37 ° C and 5% CO ₂ environment .

Experimental group cells (PLL) were prepared from mesenchymal stem cells isolated from human embryonic stem cells (H9 hESc) and cultured for 12 hours in 37 ° C, 5% CO ₂ environment using mesenchymal stem cell culture medium. And cultured in a culture vessel.

The control cells (control) are mesenchymal stem cells isolated from human embryonic stem cells (H9 hESc), and the extracellular matrix is applied using mesenchymal stem cell culture medium for 12 hours at 37 ° C and 5% CO ₂ environment Lt; / RTI &gt; culture medium.

Microscopic photographs of experimental cells (Fn, Gelatin, PLL) and control cells (Control) are shown in FIG. 1, and FIG. 2 is a graph showing quantitatively the cell adhesion rate thereof. To determine the characteristics of mesenchymal stem cells in experimental cells (Fn, Gelatin, PLL) and control cells (Control), flow cytometry was performed using a fluorescence activated cell sorter (FACS). PE (phycoerythrin) -labeled anti-CD105, APC (allo-phycocyanin) -labeled anti-CD90 antibody was used. The results are shown in FIG.

1 to 3, compared with the control cells (Gelatin, PLL), the test cells (Fn) had a significantly higher cell adhesion rate, and the surface markers of the mesenchymal stem cells (CD90 , CD105) were the most highly expressed, indicating strong characteristics of mesenchymal stem cells.

On the other hand, it was confirmed that the test cell (Fn) was attached to the fibronectin-culture vessel in the form of a fibroblast, which is a morphological characteristic of mesenchymal stem cells.

< Experimental Example  2. Each different The extracellular matrix  Cultured in an applied culture container Of mesenchymal stem cells  Changes in Focal Molecule Molecules>

Actin staining for confirming the cytoskeleton and vinculin staining for the intracellular local adhesion molecule was performed on the experimental group cells (Fn, Gelatin, PLL) used in Experimental Example 1 and control cells (Control) And the results were confirmed by confocal microscopy. Referring to FIG. 4, in the test cell (Fn), the most frequent was vinculin staining and actin staining.

Integrin expression of the test cell (Fn) used in Experimental Example 1 was confirmed by real-time PCR. RNA was extracted from the experimental cells (Fn) using the TRIzol method, samples were prepared by adding Power SYBR Green PCR Master Mix (Applied Biosystems) to the cDNA obtained using SuperScript ^TM III (Invitrogen) Were used to perform real-time PCR. The primers used for confirming integrin expression are shown in Table 2 below.

Marker Sequences (5 '-> 3') NCBI No. Integrin α1 sense (SEQ ID NO: 1)
AAT TGG CTC TAG TCA CCA TTG TT NM_181501.1 Antisense (SEQ ID NO: 2)
CAA ATG AAG CTG CTG ACT GGT Integrin α4 sense (SEQ ID NO: 3)
GAT GAA AAT GAG CCT GAA ACG NM_000885.5 Antisense (SEQ ID NO: 4)
GCC ATA CTA TTG CCA GTG TTGA Integrin α5 sense (SEQ ID NO: 5)
TGC AGT GTG AGG CTG TGT ACA NM_002205.2 Antisense (SEQ ID NO: 6)
GTG GCC ACC TGA CGC TCT Integrin αV sense (SEQ ID NO: 7)
GCA CCC TCC TTC TGA TCC T NM_001144999.2 Antisense (SEQ ID NO: 8)
GAG GAC CTG CCC TCC TTC Integrin β1 sense (SEQ ID NO: 9)
GAA GGG TTG CCC TCC AGA NM_002211.3 Antisense (SEQ ID NO: 10)
GCT TGA GCT TCT CTG CTG TT

5, integrin? 1, integrin? 5 and integrin? V were most expressed in experimental cells (Fn) except integrin? 4, and integrin? 5 was most expressed in experimental cells (Fn). Compared to control cells, integrin αV showed a high expression rate in all of the experimental cells (Gelatin, PLL, Fn), whereas integrin β1 showed low expression in all of the experimental cells (Gelatin, PLL, Fn).

In addition, an inhibition test for cell adhesion was performed using an integrin antibody. For the inhibition test on the cell adhesion, the test group cells (Fn) used in Experimental Example 1 were detached from the fibronectin-culture container, and then the integrin antibodies were treated and adhered again to the fibronectin-culture container.

6 and 7, the cell adhesion rate of mesenchymal stem cells to fibronectin was significantly lower in the experimental group (Fn) when the integrin? 5? 1 antibody was added compared to the integrin? 5 antibody and the integrin? lost.

<Example 4>

The mesenchymal stem cells obtained in Example 1 (3) were transferred to a new fibronectin-culture vessel, and the mesenchymal stem cell culture medium (10% fetal bovine serum and 1% penicillin / streptomycin (P / S) Ml DMEM-H), the first subculture was performed for 5 days under the conditions of 37, 5% CO ₂ . Subsequently, subcultures from the 2nd to 10th pass were subcultured every 5 days using a general culture container.

&Lt; Comparative Example 2 &

The fat-derived mesenchymal stem cells obtained from a 72 year old female were cultured at 37 ° C in mesenchymal stem cell culture medium (DMEM-H supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (P / S) , 5% CO ₂ , and then subcultured to the 10th passage every 5 days using a general culture vessel.

&Lt; Comparative Example 3 &

Bone marrow-derived mesenchymal stem cells (cell viroid, CB-BMMSC-005) were cultured in DMEM-H supplemented with mesenchymal stem cell culture medium (10% fetal bovine serum and 1% penicillin / ), Subcultured to the 10th passage every 5 days using a general culture vessel under the conditions of 37 ° C and 5% CO ₂ .

< Experimental Example  3. Experimental group  cell( Fn -separated MSC )of Of mesenchymal stem cells  Characteristic Analysis>

Comparison with the mesenchymal stem cells (Fn-separated MSC) obtained in Example 4 and the fat-derived mesenchymal stem cells obtained in Comparative Example 2 (control cells: ASC) using the fluorescence activated cell sorter (FACS) The characteristics of mesenchymal stem cells were analyzed for bone marrow-derived mesenchymal stem cells obtained in Example 3 (control cells: BM-MSC). All of the experimental cells (Fn-separated MSC) and control cells (ASC, BM-MSC) were cells in the third passage.

CD34 (hematopoietic stem cell marker), HLA-DR (immune rejection related marker), CD44 (mesenchymal stem cell marker), CD90 (hematopoietic stem cell marker) Stem cell marker) and CD105 (mesenchymal stem cell marker) were used as antibodies. The results are shown in Fig. 8, and the mesenchymal stem cell markers CD44, CD90 and CD105 were highly expressed, and OCT4, CD31, CD34 and HLA-DR were almost not expressed.

<Experimental Example 4> Analysis of the ability of the test cell (ES-Fn-MSC)

Analysis of adipocyte differentiation ability

Derived mesenchymal stem cells (control cell: ASC) obtained in Comparative Example 2 and the bone marrow-derived mesenchymal stem cells obtained in Comparative Example 3 (ES-Fn-MSC) The ability of mesenchymal stem cells to differentiate into adipocytes was analyzed for cells (control cells: BM-MSC). The cells in the experimental group (ES-Fn-MSC) and the control cells (ASC, BM-MSC) were all in the third passage.

Experimental cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were cultured for 14 days in a StemPro Adipogenesis Differentiation Kit (Gibco BRL). The degree of adipocyte differentiation was confirmed by the method of staining with Oil Red O staining method.

Oil red O staining was performed according to the method of Ramirez-Zacarias et al. [Histochemistry 97, 493-497 (1992)]. The test cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were washed twice with PBS and 0.5 mL of Oil red O solution (0.36% Oil red O in 60% isopropanol) , Treated at room temperature for 15 minutes, then the dye was removed and washed three times with 60% isopropanol. The results are shown in Fig.

After oil red O staining and microscopic observation, the experimental cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were washed twice with PBS and treated with 60% isopropanol, The absorbance was measured at the wavelength of nm. The results are shown in Fig.

Fattype marker analysis

On the other hand, the expression of lipid differentiation markers (PPARr, C / EBPb) was measured using real-time PCR. The cells were cultured in ES cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) in a stem cell culture medium (DMEM medium containing 1% P / S and 10% FBS) Real-time PCR was performed. RNA was extracted from the experimental cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) by the TRIzol method, and the obtained SYBR Green PCR Master Mix (SuperScript ^™ III (Invitrogen) Applied Biosystems) to prepare samples, and real-time PCR was performed using a StepOnePlus (Invitrogen) instrument. The primer sets used for real-time PCR and the respective differentiation markers are shown in Table 3 below.

Marker Sequences (5 '-> 3') NCBI No. PPARγ sense (SEQ ID NO: 11)
GAT ACA CTG TCT GCA AAC ATA TCA CAA NM_015869.4 Antisense (SEQ ID NO: 12)
CCA CGG AGC TGA TCC CAA C / EBP? sense (SEQ ID NO: 13)
GCA AGA GCC GCG ACA AG NM_005194.2 Antisense (SEQ ID NO: 14)
GGC TCG GGC AGC TGC TT GAPDH sense (SEQ ID NO: 15)
ACA TCG CTC AGA CAC CAT G NM_002046 Antisense (SEQ ID NO: 16)
TGT AGT TGA GGT CAA TGA AGG G

EXPERIMENTAL EXAMPLE 5: Analysis of the ability of ES cell-derived cells (ES-Fn-MSC)

Analysis of bone cell differentiation ability

Derived mesenchymal stem cells (control cell: ASC) obtained in Comparative Example 2 and the bone marrow-derived mesenchymal stem cells obtained in Comparative Example 3 (ES-Fn-MSC) The ability of mesenchymal stem cells to differentiate into osteocytes was analyzed for cells (control cells: BM-MSC). The cells in the experimental group (ES-Fn-MSC) and the control cells (ASC, BM-MSC) were all in the third passage.

Experimental cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were cultured in osteogenic differentiation medium (10% FBS, 100 nM dexamethasone, 1% Glutamax (Invitrogen, mu] g / mL ascorbic acid, 10 mM Glycerol 2-Phosphate, 1% P / S) for 21 days. The degree of osteoclast differentiation was confirmed by histological staining method, Alizarin red staining method.

Alizarin red staining was performed according to the method of Carl A. Gregory et al. [Analytical Biochemistry 329, 7784, (2004)]. The test cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were washed twice with PBS and 0.5 mL of 2% Alizarin red solution (2% Alizarin Red in ultrapure water) After treatment at room temperature for 30 minutes, the dye was removed and washed three times with PBS. The results are shown in Fig.

After Alizarin red staining and microscopic observation, the absorbance of the test cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) was measured at 570 nm wavelength. The results are shown in Fig.

Analysis of bone marrow markers

Meanwhile, bone differentiation markers (Col1, ALP) were analyzed by real-time PCR in the same manner as Experimental Example 4. The primer sets used for real-time PCR and the respective differentiation markers are shown in Table 4 below.

Marker Sequences (5 '-> 3') NCBI No. Col1 sense (SEQ ID NO: 17)
CAC GTA CAC TGC CCT GAA GGA NM_001844 Antisense (SEQ ID NO: 18)
CGA TAA CAG TCT TGC CCC ACT T ALP sense (SEQ ID NO: 19)
GAC AAG AAG CCC TTC ACT GC NM_000478 Antisense (SEQ ID NO: 20)
AGA CTG CGC CTG GTA GTT GT GAPDH sense (SEQ ID NO: 15)
ACA TCG CTC AGA CAC CAT G NM_002046 Antisense (SEQ ID NO: 16)
TGT AGT TGA GGT CAA TGA AGG G

EXPERIMENTAL EXAMPLE 6. Analysis of the ability of ES cells (ES-Fn-MSC) to differentiate into chondrocytes.

Analysis of cartilage cell differentiation ability

Derived mesenchymal stem cells (control cell: ASC) obtained in Comparative Example 2 and the bone marrow-derived mesenchymal stem cells obtained in Comparative Example 3 (ES-Fn-MSC) (Control cells: BM-MSC) were analyzed for the ability of mesenchymal stem cells to differentiate into chondrocytes. The cells in the experimental group (ES-Fn-MSC) and the control cells (ASC, BM-MSC) were all in the third passage.

(10% FBS, 100 nM dexamethasone, 50 μg / mL ascorbic acid, 1% P / S, 1% FBS) were added to the chondrocyte differentiation medium (ES-Fn-MSC) and control cells , DMEM medium containing ITS (insulin 1 g / L, sodium selenite 0.67 mg / L, transferrin 0.5 g / L), 10 ng / mL TGF-β1) for 21 days. The degree of chondrocyte differentiation was confirmed by Alcian blue staining method. Experimental cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were washed twice with PBS, placed in 4% Alcian blue solution and treated at room temperature for 30 minutes. The results are shown in Fig.

After the Alcian blue staining and microscopic observation, the experimental cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were reacted with 6M guanidine-HCl at room temperature for 2 hours, Absorbance was measured. The results are shown in Fig.

Analysis of cartilage differentiation markers

Meanwhile, the cartilage differentiation markers (Col2, AGG) were analyzed by real-time PCR in the same manner as in Experimental Example 4. The primer sets used in the real-time PCR and the respective differentiation markers are shown in Table 5 below.

Marker order NCBI No. Col2 sense (SEQ ID NO: 21)
5'-TTC AGC TAT GGA GAT GAC AAT C-3 ' NM_001844 Antisense (SEQ ID NO: 22)
5'-AGA GTC GAG TGA CTG AG-3 ' AGG sense (SEQ ID NO: 23)
5'-GAA TCT AGC AGT GAG ACG TC-3 ' NM_013227 Antisense (SEQ ID NO: 24)
5'-CTG CAG CAG TTG ATT CTG AT-3 ' GAPDH sense (SEQ ID NO: 15)
5'-CGG ATT TGG TCG TAT TGG GC-3 ' NM_002046 Antisense (SEQ ID NO: 16)
5'-CAG GGA TGA TGT TCT GGA GA-3 '

< Experimental Example  7. Experimental group  cell( ES - Fn - MSC ) And analysis of telomere length change>

Cell survival analysis

The cell viability of the mesenchymal stem cells (experimental cell: ES-Fn-MSC) obtained in Example 4 was confirmed using a live / dead assay kit. Experimental cells (ES-Fn-MSC) were cells obtained in the third passage. Experimental cells (ES-Fn-MSC) were washed three times with PBS and stained with 4 mM calcein AM (Invitrogen) and 4 μM Ethidium Homodimer (EthD-1, Invitrogen) , And live cells (green) and dead cells (red) were observed with a confocal laser scanning microscope (CLSM). The results are shown in FIG. All cells showed green fluorescence, and no cells with red fluorescence were found.

Cell proliferation capacity analysis

MSCs) obtained in Example 4 and the fat-derived mesenchymal stem cells (control cells: ASC) obtained in Comparative Example 2 were compared with each other using a hematocytometer MSC) of bone marrow-derived mesenchymal stem cells obtained in Example 3 (control cells: BM-MSC). Cells from experimental group (ES-Fn-MSC) and control cells (ASC, BM-MSC) were obtained from each passage (passage 3 to passage 10).

Experimental cells (ES-Fn-MSC) and control cells (ASC, BM-MSC) were cultured for 5 days, and each cell was washed once with DPBS and made into single cells using trypsin. Each cell was counted using a hemocytometer. The results are shown in Fig.

Cell cycle analysis

Derived mesenchymal stem cells (control cells: ES-Fn-MSC (p3)) obtained in Example 4 and the fat-derived mesenchymal stem cells obtained in Comparative Example 2 (control cells: ASC (p3)) and bone marrow-derived mesenchymal stem cells obtained in Comparative Example 3 (control cells: BM-MSC (p3)). The cells in the experimental group (ES-Fn-MSC (p3)) and control cells (ASC (p3) and BM-MSC (p3)

MSC (p3)) and control cells (ASC (p3), BM-MSC (p3)) were harvested and centrifuged, fixed with 70% cold ethanol, Immobilized cells were centrifuged and washed with PBS. The cell pellet was suspended in a Propidium Iodide (PI) staining solution (PBS supplemented with 0.1% Triton X-100, 20 g / mL PI and 0.2 mg / mL RNase) and reacted at 37 ° C for 15 minutes . The samples were then analyzed. The results are shown in Fig. 17 and it was confirmed that the number of experimental cells (ES-Fn-MSC (p3)) in S phase was larger than that of control cells (ASC (p3) and BM-MSC (p3)

Telomere length measurement

derived mesenchymal stem cells (control cells: ASC) obtained in Comparative Example 2 and Comparative Example 3 (control cells: ASCs) obtained in Example 4 using real-time PCR, (Control cells: BM-MSC) obtained from the bone marrow-derived mesenchymal stem cells. The cells in the experimental group (ES-Fn-MSC) and the control cells (ASC, BM-MSC) were all in the third passage. The primer sets and the respective markers used in the real-time PCR are shown in Table 6 below.

Marker order 36B4u / d u (SEQ ID NO: 25)
5'-CAG CAG GTG GGA AGG TGT AAT CC -3 ' d (SEQ ID NO: 26)
5'-CCC ATT CTA TCA TCA ACG GGT ACAA -3 ' tel1 / 2 One (SEQ ID NO: 27)
5'-GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA GGG T-3 ' 2 (SEQ ID NO: 28)
5'- TCC CGA CTA TCC CTA TCC CTA TCC CTA TCC CTA TCC CTA -3 '

RNA was extracted from the experimental cells (ES-Fn-MSC) and control cells (BM-MSC, ASC) by the TRIzol method and the obtained SYBR Green PCR Master Mix (SuperScript ^™ III, Invitrogen) Applied Biosystems) to prepare samples, and real-time PCR was performed using a StepOnePlus (Invitrogen) instrument. The results are shown in Fig.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It is to be understood that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Colleage of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> SORTING METHOD OF MESENCHYMAL STEM CELL DERIVED FROM PLURIPOTENT          STEM CELL <130> C28-61020 <160> 28 <170> KoPatentin 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha 1 sense <400> 1 aattggctct agtcaccatt gtt 23 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha 1 antisense <400> 2 caaatgaagc tgctgactgg t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha 4 sense <400> 3 gatgaaaatg agcctgaaac g 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha 4 antisense <400> 4 gccatactat tgccagtgtt ga 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha 5 sense <400> 5 tgcagtgtga ggctgtgtac a 21 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha 5 antisense <400> 6 gtggccacct gacgctct 18 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha V sense <400> 7 gcaccctcct tctgatcct 19 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> integrin alpha V antisense <400> 8 gaggacctgc cctccttc 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> integrin beta 1 sense <400> 9 gaagggttgc cctccaga 18 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> integrin beta 1 antisense <400> 10 gcttgagctt ctctgctgtt 20 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma sense <400> 11 gatacactgt ctgcaaacat atcacaa 27 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma antisense <400> 12 ccacggagct gatcccaa 18 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> C / EBP beta sense <400> 13 gcaagagccg cgacaag 17 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> C / EBP beta antisense <400> 14 ggctcgggca gctgctt 17 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense <400> 15 acatcgctca gacaccatg 19 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense <400> 16 tgtagttgag gtcaatgaag gg 22 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Col1 sense <400> 17 cacgtacact gccctgaagg a 21 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Col1 antisense <400> 18 cgataacagt cttgccccac tt 22 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALP sense <400> 19 gacaagaagc ccttcactgc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALP antisense <400> 20 agactgcgcc tggtagttgt 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Col2 sense <400> 21 ttcagctatg gagatgacaa tc 22 <210> 22 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Col2 antisense <400> 22 agagtcgagt gactgag 17 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AGG sense <400> 23 gaatctagca gtgagacgtc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AGG antisense <400> 24 ctgcagcagt tgattctgat 20 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 36B4u <400> 25 cagcaagtgg gaaggtgtaa tcc 23 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 36B4d <400> 26 cccattctat catcaacggg tacaa 25 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> tel1 <400> 27 ggtttttgg ggtgagggtg agggtgaggg tgagggt 37 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> tel2 <400> 28 tcccgactat ccctatccct atccctatcc ctatcccta 39

Claims (15)

Spontaneous differentiation of pluripotent stem cells into differentiated cells including ectodermal, mesodermal, and endodermal cells;
Culturing the differentiated cells in a fibronectin_coated culture container using mesenchymal stem cell culture medium; And
And naturally selectively separating the mesenchymal stem cells attached to the fibronectin culture container,
The mesenchymal stem cells are selected from pluripotent mesenchymal stem cells derived from pluripotent mesenchymal stem cells, which exhibit positive immunological characteristics for both CD90 and CD105 and satisfy the following formula 1:
<Formula 1>

In Equation (1)
CD105 (Fn) is the expression rate of CD105 in the mesenchymal stem cell attached to the fibronectin, and CD105 (G) is the expression rate of CD105 in the mesenchymal stem cell attached to the gelatin. The method according to claim 1,
Further comprising culturing the pluripotent stem cells in a feeder cell-free environment, wherein the pluripotent stem cells are cultured in a feeder cell-free environment. The method according to claim 1,
Wherein said mesenchymal stem cells are selected from pluripotent stem cell-derived mesenchymal stem cells adhered to said fibronectin-cultivation vessel via integrin? 5? 1. delete The method according to claim 1,
The mesenchymal stem cells are selected from pluripotent mesenchymal stem cells derived from pluripotent mesenchymal stem cells, which exhibit positive immunological characteristics for both CD90 and CD105 and satisfy the following formula 2:
<Formula 2>

In the above formula 2,
CD105 (Fn) is the expression rate of CD105 in the mesenchymal stem cell attached to the fibronectin (D) culture vessel, and CD105 (PLL) is the expression rate of CD105 in the mesenchymal stem cell attached to the poly-L- . The method according to claim 1,
Wherein the mesenchymal stem cells are attached to the fibronectin in a fibroblast-like manner. The method according to claim 1,
Wherein said pluripotent stem cells are selected from the group consisting of human embryonic stem cells, mesenchymal stem cells derived from pluripotent stem cells. The method according to claim 1,
Wherein the pluripotent stem cell is a human induced pluripotent stem cell, the pluripotent stem cell derived from an allogeneic stem cell. The method according to claim 1,
Wherein said mesenchymal stem cells express immunologic characteristics that are all positive for CD44, CD90 and CD105, from mesenchymal stem cells derived from pluripotent stem cells. Spontaneous differentiation of pluripotent stem cells into differentiated cells including ectodermal, mesodermal, and endodermal cells;
Culturing the differentiated cells in a fibronectin_coated culture container using mesenchymal stem cell culture medium; And
And naturally selectively separating the mesenchymal stem cells attached to the fibronectin culture container,
Wherein said mesenchymal stem cells are selected from pluripotent mesenchymal stem cells derived from pluripotent mesenchymal stem cells, which have a high ability to differentiate into adipocytes as compared to bone marrow-derived mesenchymal stem cells. 11. The method of claim 10,
Wherein said mesenchymal stem cells are selected from pluripotent stem cell-derived mesenchymal stem cells, wherein PPARγ and C / EBPβ are highly expressed compared to fat-derived mesenchymal stem cells. Spontaneous differentiation of pluripotent stem cells into differentiated cells including ectodermal, mesodermal, and endodermal cells;
Culturing the differentiated cells in a fibronectin_coated culture container using mesenchymal stem cell culture medium; And
And naturally selectively separating the mesenchymal stem cells attached to the fibronectin culture container,
Wherein said mesenchymal stem cells are selected from pluripotent mesenchymal stem cells derived from pluripotent mesenchymal stem cells having osteoclast differentiation potential and chondrocyte hyperdifferentiating ability compared to bone marrow-derived mesenchymal stem cells. 13. The method of claim 12,
Wherein said mesenchymal stem cells are selected from pluripotent stem cell-derived mesenchymal stem cells, wherein Col1, ALP, Col2 and AGG are highly expressed compared to bone marrow-derived mesenchymal stem cells. Spontaneous differentiation of pluripotent stem cells into differentiated cells including ectodermal, mesodermal, and endodermal cells;
Culturing the differentiated cells in a fibronectin_coated culture container using mesenchymal stem cell culture medium; And
And naturally selectively separating the mesenchymal stem cells attached to the fibronectin culture container,
Wherein said mesenchymal stem cells are selected from the group consisting of pluripotent mesenchymal stem cells derived from pluripotent stem cells,
<Formula 3>
T / S (ASC) < T / S (ES-Fn-MSC)
In Equation (3)
T / S (ASC) is the telomere length of fat-derived mesenchymal stem cells, and T / S (ES-Fn-MSC) is the telomere length of mesenchymal stem cells. Spontaneous differentiation of pluripotent stem cells into differentiated cells including ectodermal, mesodermal, and endodermal cells;
Culturing the differentiated cells in a fibronectin_coated culture container using mesenchymal stem cell culture medium; And
And naturally selectively separating the mesenchymal stem cells attached to the fibronectin culture container,
Wherein the mesenchymal stem cells are selected from the group consisting of pluripotent mesenchymal stem cells derived from pluripotent stem cells,
<Formula 4>
T / S (BM-MSC) < T / S (ES-Fn-MSC)
In Equation (4)
T / S (BM-MSC) is the telomere length of bone marrow-derived mesenchymal stem cells, and T / S (ES-Fn-MSC) is the telomere length of mesenchymal stem cells.

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