WO2011126264A2 - Method for increasing activity in human stem cell - Google Patents

Abstract

Provided are a method for preparing a highly active human mesenchymal stem cell, which includes forming a spherical cell aggregate by cultivating human mesenchymal stem cells against gravity; a highly active stem cell aggregate prepared thereby; a cell theraputic including the stem cell aggregate; and a method for forming a spherical cell aggregate by cultivating human mesenchymal stem cells, wherein the amount of E-cadherin in the mesenchymal stem cell is increased during the cultivation.

Description

 How to increase the activity of human stem cells

FIELD OF THE INVENTION The present invention relates to a method for producing a highly active human mesenchymal stem cell mass, a highly active stem cell mass induced by the method, and a cell therapeutic agent comprising the stem cell mass. Background Stem cells are cells capable of differentiating into various cells constituting biological tissues, which collectively refer to the undifferentiated cells obtained from each tissue of the embryo, fetus and adult. Stem cells are differentiated into specific cells by differentiation stimulation (environment), and unlike the cells in which the differentiation is completed and cell division is stopped, they proliferate because they can produce the same cells as themselves by cell division. It can be differentiated into other cells by different environment or differentiation stimulus, so it has plasticity in differentiation.

 Stem cells can be divided into pluripotency, multipotency and unipotency stem cells according to their differentiation ability. Pluripotent stem cells are pluripotency cells that have the potential to differentiate into all cells. Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS) ) And the like. Multipotent and / or unipotent stem cells include adult stem cells.

Embryonic stem cells are formed from the i 皿 er cell mass of the blastocytes, which are the early stages of embryonic development, and have the potential to differentiate into all cells, which can differentiate into any tissue cell and do not die. It can be cultured in undifferentiated state, and unlike adult stem cells, it is also possible to manufacture germ cells, so it can be inherited to the next generation. (Thomson et al., Science, 282: 1145-1147 (1998); Reubinoff et al., Nat. Biotechno L, 18: 399-404 (2000)).

 Human embryonic stem cells are produced by separating and culturing only intracellular masses when forming human embryos. Currently, human embryonic stem cells made worldwide are obtained from the remaining embryonic embryos after infertility. Various attempts have been made to use human embryonic stem cells having pluripotency capable of differentiating into all cells as cell therapy, but have not completely solved problems such as cancer risk and immune rejection. Recently, as a complementary measure, iPS has been reported. IPS is a cell that has redifferentiated differentiated somatic cells by various methods and reverted to the state of embryonic stem cells, which is an early stage of differentiation. To date, iPS has been reported to have almost the same characteristics as embryonic stem cells, pluripotent stem cells, in gene expression and differentiation ability. In the case of iPS, the risk of immune rejection reaction can be eliminated by using autologous cells, but the risk of cancer still remains to be solved.

 Recently, as an alternative for overcoming these problems, mesenchymal stem cells have been proposed, along with immunomodulatory functions, without the risk of cancer. Mesenchymal stem cells are pluripotent cells capable of differentiating into adipocytes, bone cells, chondrocytes, muscle cells, nerve cells, cardiomyocytes, hepatocytes, pancreatic beta cells, and vascular cells. It is known to have.

Mesenchymal stem cells can be isolated and cultured in various tissues such as bone marrow, umbilical cord blood, and adipose tissue, but it is not easy to clearly define mesenchymal stem cells because the cell surface markers of each origin are slightly different. However, they can differentiate into osteocytes, chondrocytes, and muscle cells, have a swirl shape, and express the basic cell surface markers CD73 (+), CD105C +), CD34 (-), and CD45 (-). It is generally defined as mesenchymal stem cells. In this regard, mesenchymal stem cells of different genetic origins and / or backgrounds, although not significantly different from each other in respect of the criteria for defining the existing mesenchymal stem cells mentioned above, are typically There is a significant difference in activity in vivo. It is also derived from other stem cells When used as a cell therapy, there is a limited pool of candidates, so even if the in vivo activity of the mesenchymal stem cells is low, there is no choice and replacement is impossible.

 In addition, mesenchymal stem cells generally require a minimum number of cells required for regenerative medicine and / or cell therapy in order to be used as cell therapy.

1x10 ⁹ ), but if you consider the experiments to determine the appropriate conditions and set the standard, the number of cells required will be further increased. Therefore, supplying this amount from existing mesenchymal stem cells of various origins requires at least 10 passages in vitro in vitro, in which case the cells are aged and modified and no longer suitable for cell therapy. The problem arises.

 Therefore, the mesenchymal stem cells, which are expected to be the most desirable materials for cell therapy, can be efficiently used for the purpose of cell therapy. It is necessary to develop a solution that can be used. SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing highly active stem cell mass from mesenchymal enjoyment cells which are aged or have relatively low in vivo activity.

 It is another object of the present invention to provide a highly active mesenchymal stem cell aggregate produced by the above method and a cell therapy comprising the same. In accordance with the above object, the present invention provides a method for producing highly active human mesenchymal stem cell mass, comprising culturing human mesenchymal stem cell against gravity to form a globular cell mass.

The present invention also provides a highly active mesenchymal stem cell mass prepared by the above method and a cell therapy comprising the stem cell mass. In another aspect, the present invention comprises culturing human mesenchymal stem cells to form globular cell masses, characterized in that to increase the amount of E-cadherin in mesenchymal stem cells during the culture, It provides a manufacturing method. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects and features of the present invention will become apparent from the following description of the invention in conjunction with the accompanying drawings, in which: Figure 1 is a human mesenchymal stem using a medium in which bFGF has been removed from a human embryonic stem cell culture medium; Incubation of the cells indicates that the suspension is induced.

 Figure 2 shows the result of inducing spheroid formation by floating culture of human mesenchymal stem cells in a low adhesion dish (dish).

 Figure 3 shows the results of inducing spheroid culture by floating culture of mesenchymal stem cells against gravity in a culture dish lid.

 Figure 4 shows the improvement of ischemic heart disease by quantifying the left ventricular end-diastolic dimension (LVE DD) and left ventricular end-systolic dimension (LVESD).

 FIG. 5 shows the improvement of ischemic heart disease by quantifying left ventricular end-Ejection Fraction (LVEF) and left ventricular end-fractional shortening (LVFS).

Figure 6 shows the improvement of ischemic heart disease by quantifying the infarcted wall thickness and the infarct area, respectively. FIG. 7 is a diagram showing that the spheroid group injected with the mesenchymal stem cells that did not induce the formation of the globular cell mass, compared to the naive group injected with the group (naive), showed a significantly higher number of cells remaining near the ischemic heart. .

 Figure 8 is a picture confirming the expression of sacomeric actinin (Fig. 8a) and connexin 43 (Fig. 8b) in the spherocytic injection group.

 9 shows the vascular specific marker isolectin B4 expression in order to examine the effect on angiogenesis and quantified the result.

 Figure 10 is the result of confirming the expression of Isolectin B4 to see whether the injection of mesenchymal stem cells into vascular cells.

11 shows the result that no sphere is formed when EDTA is added. FIG. 12 shows the results of Western blot changes in the formation process of Ca ^{+ 2} dependent cell adhesion factors, N-cadherin and E-cadherin. Figure 13 shows the effect on the formation of mesenchymal stem cells when the function of E-cadherin is inhibited.

 Figure 14 shows the effect on the spheroid formation using an adenoviral vector (E— cadherin adenoviral vector) overexpressing E-cadherin.

 Figure 15 shows the change in activity of extracellular signal-regulated kinase (ERK) and AKT (V-akt murine thymoma viral oncogene homolog) according to the embodiment.

 16 shows the effect of E-cadherin on the activity of ERK and AKT.

 Figure 17 shows the results of the changes in the activity of ERK and AKT using an adenoviral vector (E- cadherin adenoviral vector) overexpressing E- cadherin.

 Figure 18 shows the effect of E-cadherin on the cell growth of mesenchymal stem cells.

19 shows the effect of E-cadherin on cell death of mesenchymal stem cells. Figure 20 shows the effect of E-cadherin on the secretory capacity of VEGF (Vascular endothelial growth factor, vascular endothelial growth factor) of mesenchymal stem cells.

 Figure 21 shows the results of the MLR (mixed lymphocyte reaction) to evaluate the degree of immunity in vitro using two cord blood-derived mesenchymal stem cells (UCB-MSC) of different origin.

22 is a result of analyzing the PGE ₂ secretion level in each MLR culture of FIG. 21 by ELISA method.

 Figure 23 is a result of examining the effect of specificity on immune function using F4 / 80, a marker of immune cells.

 24A is a survival / death staining result for confirming the inhibitory effect of cartilage cell death due to spheroid formation of mesenchymal stem cells, and FIG. 24B is a graph showing survival rate therefrom.

 25 is a result of visual analysis and tissue staining analysis of the cartilage damage site after 10 weeks in the rabbit cartilage defect model to confirm the cartilage regeneration effect due to the spheroid formation of mesenchymal stem cells.

 FIG. 26 compares the expression changes of differentiation activators in pulmonary cell differentiation in spheroids made by the hanging drop method and the bioreactor method. Detailed Description of the Invention The present invention provides a method for producing highly active human mesenchymal stem cell mass. Specifically, the present invention provides a method for producing highly active human mesenchymal stem cell mass, comprising culturing human mesenchymal stem cells against gravity to form globular cell mass.

 As used herein, "stem cell mass", "aggregate" or "sphere" means a spherical aggregate of stem cells formed by culturing stem cells, and are used interchangeably.

Human mesenchymal stem cells used in the present invention is not limited in its genetic background and / or origin. For example, human cord blood-derived mesenchyme Stem cells, adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells can be used, umbilical cord blood-derived mesenchymal stem cells are preferred. In the present invention, the culture for the formation of the globular cell mass may be to culture the mesenchymal stem cells in a drop of medium placed against gravity. At this time, it is advantageous to obtain a spherical cell mass having high therapeutic activity by including 300 to 30,000 cells, preferably 1,000 to 30,000 cells per medium drop to form a globular cell mass therefrom.

 According to the method for culturing stem cells against gravity as described above, since a large number of stem cell masses of uniform size can be obtained, there is an advantage of increasing therapeutic effectiveness by using them.

 In the method of the present invention, as a culture medium, serum excrement medium containing serum replacement (SR) may be used. As SR, any commercially available can be used, the concentration of SR in the medium can be adjusted as needed, and 20¾ (v / v) is preferred.

 The serum exclusion medium may be human embryonic stem cell culture medium that does not include serum and basic fibroblast growth factor (bFGF). The present invention also includes culturing human mesenchymal stem cells to form globular cell masses, characterized in that to increase the amount of E-cadherin in the mesenchymal stem cells, the highly active human mesenchymal stem cell masses It provides a method for producing.

 An increase in the amount of E-cadherin in the stem cells can be achieved by introducing a vector expressing E-cadherin in the mesenchymal stem cells. The expression vector may be, for example, an adenovirus vector containing an E-cadherin gene.

In this method, the culturing of mesenchymal stem cells to form globular cell mass may be performed by culturing against gravity using a medium as described above or by floating culture in a low adhesion culture dish. In the case of floating culture, the method may further include separating the generated globular cell mass and cells not included in the globular cell mass. In this separation step, Any tool capable of separating the cell mass and the single cell may be used, preferably using a strainer.

 Alternatively, the globular cell mass may be obtained by culturing the mesenchymal stem cells in a medium as described above using a three-dimensional bioreactor (spire) or by agitating in a common adherent container. Can be formed by reducing the chance of attachment to the bottom or by culturing single cells under conditions such as stress, for example hypoxia or low temperature below room temperature; Place a number of stem cells in a plate with a fine well structure at the bottom, such as a product called AggreWel ™, or place single cells in a non-adherent container or stem cell therapy device that is difficult to attach It can also naturally form globular cell mass. The present invention also provides a highly active human mesenchymal stem cell mass produced by the above production method.

 Stem cell mass of the present invention is excellent in tissue regeneration and disease treatment effect when administered in vivo, has a high survival rate in vivo, and has the advantage of high efficiency of differentiation into tissue cells. In another aspect, the present invention provides a cell therapy agent comprising the highly active human mesenchymal stem cell mass.

 The cell therapy agent of the present invention can be used for the formation of adipocytes, bone cells, chondrocytes, muscle cells, nerve cells, cardiomyocytes, hepatocytes, pancreatic beta cells, vascular cells or lung cells.

 In addition, the cell therapy of the present invention; Inhibiting or treating inflammation caused by lung disease; Lung tissue regeneration; And it is useful for any one selected from the group consisting of pulmonary tissue fibrosis inhibitory, it can suppress or improve the inflammatory reaction and fibrosis (f ibrosis) caused by lung disease.

The cell therapy of the present invention can be used for the treatment of cardiovascular diseases or for cartilage regeneration. In addition, the cell therapy of the present invention may increase immunomodulatory function, lower immunostimulatory, immune cell infiltration or immunogenicity, and inhibit inflammatory response. In addition, the present invention provides a method for mass-producing highly active human mesenchymal stem cells using a bioreactor.

 Bioreactors are systems or equipment that maintain and support a biologically active environment. Highly active human mesenchyme capable of inducing spherical cell mass of human mesenchymal stem cells in the bioreactor, and further growing without contact inhibit ion if the formed spherical cell mass continues to be cultured in the bioreactor. Stem cells can be mass produced. That is, the culture medium as described above can be used to induce sphere formation by centrifugal force using agitation (st irring), etc. in the bioreactor, and then continuously cultured using the same medium, spherical high-activity human intermediate Massive amplification of lobe stem cells is possible. According to the present invention, it is possible to maximize the practicality and therapeutic efficiency of mesenchymal stem cells as a cell therapeutic agent by increasing the activity of senile mesenchymal stem cells which are aging or relatively low in vivo. In addition, the high activity induction method of the present invention is a standardized method that can be applied to various human mesenchymal stem cells having different genetic backgrounds and / or origins, and can be very useful for developing and selecting a taga-derived cell therapy. There will be.

 In addition, the present invention maximizes the efficiency of human mesenchymal enjoyment cells, thereby enabling to derive the appropriate number of highly functional human mesenchymal stem cells required for cell therapy and regenerative medicine. The present invention also enables the mass production of highly active human mesenchymal stem cells.

Ultimately, the present invention may increase the efficiency of human mesenchymal stem cells as cell therapeutics, thereby promoting the practical use of cell therapeutics, and further contribute to the development of therapeutic agents for cardiovascular diseases and nervous system diseases. It is expected. Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example In the present invention, human cord blood-derived mesenchymal stem cells provided by MEDIP0ST Co., Ltd. (Korea) were used. The cells were selected by performing human mesenchymal stem cell identification experiments, expressing at least 95% of positive cell markers (CD29, CD44, CD73, CD105, CD166, HLA-ABC) and negative cell markers (CD34) of mesenchymal stem cells. , CD45, HLA-DR) was identified and classified as "human cord blood-derived mesenchymal stem cells" after confirming the uniform expression of less than 5%, and confirming the multipotent differentiation of mesenchymal stem cells. Example 1 Induction of Sphere Formation of Human Mesenchymal Stem Cells (1) Spheroid Formation Induction Medium First, the mesenchymal stem cells were replaced with SR (serum replacement) in a-MEM (manufactured by Invitrogen), which is an existing mesenchymal stem cell culture medium. Attempted suspension culture in a low adhesion dish using the medium added (see Figure 1A).

Next, the mesenchymal stem cells were cultured in a low adhesion dish using a medium from which basic fibroblast growth factor (bFGF) was removed from embryonic stem cell media (ESM). (See FIG. 1B). The medium does not contain fetal bovine serum (bovine serum), DMEM / F-12 (Invitrogen), 20% Knock out SR (Invitrogen), O.lmmol / L -mercaptoethanol (Sigma), 1% non-essential amino acids (Invitrogen), 50 IU / ml penicillin and 50 mg / ml strapomycin (Invitrogen).

(2) Sphere Formation Method The present inventors induced sphere formation of human mesenchymal stem cells by using two methods, and spheres were successfully formed by both of the following methods.

 First, spheroid formation was induced by culturing human mesenchymal stem cells using a bFGF-free ESM medium of (1) in a low adhesion dish, and the results are shown in FIG. 2. Mesenchymal globular cell mass induced by this method was separated from cells not included in the formation of spheres using a strainer.

 As another method for inducing spherical formation, inoculating mesenchymal stem cells into culture dish lids 300-3000 eel Is / medium 20ul using the same medium as above and incubating against gravity with the lid upside down. Sphere formation was induced and the results are shown in FIG. 3. This method is capable of controlling the number of cells and uniformizing the size of the formed spheres has the advantage of obtaining a stem cell mass with high therapeutic efficacy. Therefore, all subsequent experiments were performed using stem cell mass obtained by this method except where otherwise stated. Example 2 Effect of Sphere Formation-Effect on In Vivo Activity The in vivo activity of mesenchymal stem cells was evaluated using an ischemic cardiovascular disease rat model. The ischemic cardiovascular rat model was made by inducing ischemic state by ligation of coronary artery of heart.

Ischemic cardiovascular disease rat models were injected with the spheroid cell mass itself obtained in Example (2) (spheroid), the group was injected into a single cell after formation of the globular mass (dissociate), and spherical Cell mass The experiment was conducted by dividing the cells which did not induce formation into naive groups, and at least 7 rats were used for each of the groups. (1) ECG measurement Baseline cardiac conduction was measured 4 days after the disease modeling, and stem cells were injected into the disease model on day 7 after the disease modeling. Specifically, stem cells or cell masses were injected around the myocardium in which ischemic heart disease was induced using Hamilton's Shirin, which was made of glass without friction, and the number of mesenchymal stem cells injected per rat was adjusted to 1X10 ⁵ . Left ventricular end-diastolic dimension (LVEDD), left ventricular end—systolic dimension (LVESD), and left ventricular fractional shortening after measurement of cardiac conduction at 4 and 8 weeks after the cell injection. (Left ventricular end-Fractional Shortening (LVFS)) and left ventricular blood count (Left ventricular end-Ejection Fraction (LVEF)) were quantified to analyze the improvement of the disease. Left ventricular fractional shortening (LVFS) is defined as LVEDD-LVESD / LVEDD and left ventricular blood count (LVEF) is defined as LVEDD ² -LVESDVLVEDD ² . The smaller the left ventricular diastolic ^' LVDD and the left ventricular systolic diameter (LVESD), the higher the left ventricular fractional shortening (LVFS) and the left ventricular blood cell count (LVEF).

As can be seen in Figure 4, in the case of the group injected with the spherical cell mass itself (spheroid) and the group injected with a single cell after the formation of the spherical cell mass (dissociate), the group injected with cells that did not induce spherical cell formation Left ventricular diastolic diameter (LVEDD) and left ventricular systolic diameter (LVESD) were smaller than naive. Especially, the spheroid group injected with spherical cell mass itself showed significantly lower LVEDD and LVESD values than the group injected with cells that did not induce spherical cell formation. Low LVEDD and LVESD values were also observed when compared to the dissociate injected with single cells. In addition, as can be seen in Figure 5, in the case of the group injected with the spherical cell mass itself (spheroid) and the group of dissociate injected into a single cell after the formation of the globular cell mass (dissociate), the cells that do not induce the formation of the globular cell mass were injected. Higher left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) values were shown compared to naive. Especially, the spheroid group injected with spherical cell mass itself showed significantly higher LVFS and LVEF values than the group injected with cells that did not induce spherical cell mass formation. High LVFS and LVEF values were also observed when compared with the injected group (dissociate).

 From the above results, it can be seen that when the spherical cell mass itself is injected, it exhibits high improvement.

(2) Comparison of heart size and fibrosis In addition to the measurement of cardiac conduction, we examined the effects on the overall heart wall thickness and fibrosis when injecting spherical cell mass. Figure 6 shows the numerical representation of the infarcted wall thickness and the infarct area, respectively.

 In general, when the heart is exposed to ischemia, fibrosis of the heart wall causes thinning of the heart wall, loss of motility and volume expansion. As can be seen in Figure 6, when the spheroid cell mass itself was injected, compared with the group injected with cells that did not form the globular mass, even when exposed to ischemia, the heart wall that appears as the progress of the ischemia is thinner. It can be seen that the phenomenon of fibrosis is significantly reduced. In addition, when the spherical cell mass itself was injected, the formation of the spherical cell mass and then separated into a single cell was compared with the group injected with a thinning of the wall and the progression of fibrosis was relatively reduced.

(3) histological analysis of the heart In order to elucidate the cause of in vivo activity (improvement) on the cardiac ischemia model, an experiment was performed to analyze the heart. In order to facilitate the tracking of remaining mesenchymal stem cells after injection, the cells were dil-stained and injected into the ischemia model.

 Dil (l, -dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) is a hydrophobic and lipophilic substance that attaches to the cell's double lipid membrane and marks the cells in red. . To identify the injected cells in the tissue, heart tissue was sampled,

Nuclear staining with DAPI and confirmed by fluorescence microscopy. DAPI (4 ', 6-diamidino-2-phenylindole) is a substance that strongly adheres to the minor groove of the AT cluster of double-stranded DNA of all cells and shows blue fluorescence.

 As shown in FIG. 7, in the case of the spheroid group injected with the mesenchymal stem cell which did not induce the formation of the globular cell mass, the cells remaining near the ischemic heart were significantly higher. Could confirm. In other words, the significant number of cells stained with Dil (red) means that the globular cell mass has an excellent effect in terms of survival rate of the injected cells. In addition, we confirmed the expression of sacomeric actinin (S-actinin), a myocardial specific marker, to confirm the differentiation of mesenchymal stem cells remaining near the ischemic heart into cardiomyocytes lost to ischemia. Expression of connexin 43CCX43), which plays an important role in connection with cells, was confirmed.

As can be seen in Figure 8, the expression of S-actinin (stained green part) in the globular cell infusion group was confirmed (see Figure 8a), which is the mesenchymal stem cells remaining near the ischemic heart is lost Differentiation induced into cardiomyocytes. In addition, the expression of connexin 43 (stained with green part), which is important for cardiomyocyte function, was confirmed in the globule infusion group (see FIG. 8B). In addition, an experiment was conducted to examine the effect on the most important angiogenesis in improving ischemia heart. Specifically, the expression of isolectin B4, a vascular specific marker, was confirmed and the result was quantified, and is shown in FIG. 9.

As shown in FIG. 9, isolectin B4, which is a vascular specific marker, was markedly expressed in the spheroid group injected with the globular cell mass compared to the naive group injected with the mesenchymal stem cells which did not induce the globular family. As shown in the graph quantifying the number per mm ² , it was confirmed that it showed more than twice the excellent angiogenic effect. Experiments were conducted to determine whether the difference in angiogenesis was due to the differentiation of mesenchymal stem cells into vascular cells remaining in the ischemic model. Specifically, the result of staining with isolectin B4 together with Dil was analyzed.

As shown in FIG. 10, isolectin B4 was more remarkably expressed in the spheroid group injected with the globular cell mass compared to the naive group injected with the mesenchymal stem cells which did not induce the globular cell mass. Colored part), which means that the differentiation rate of mesenchymal stem cells into vascular cells was significantly increased due to the spheroid formation. From the above results, spheroid cell cultures of human mesenchymal stem cells were formed to form spheres, and the cells that did not form spheres were injected into the ischemic heart disease model. It can be seen that it shows a markedly increased therapeutic effect compared to the single cells isolated again after the formation. In addition, the improved therapeutic effect of ischemic heart disease in the globular cell infusion group was confirmed to be due to the markedly increased cell survival rate, differentiation efficiency into cardiomyocytes, and angiogenic capacity due to spherical formation. It was. In conclusion, induction of splenic mesenchymal stem cells increased the activity of mesenchymal stem cells, and further, maintaining the formed spheres further increased the activity of mesenchymal stem cells as compared to separating them into single cells. It is obvious that the increase. Example 3: Mechanism Analysis of Sphere Formation-In Vivo Activity Difference Verification An experiment was conducted to analyze the mechanism of spherical formation causing the difference in in vivo activity described in Example 2 above.

First, in the low adhesion dish, EDTA was added to the induction of spheroid formation using bFGF-free ESM medium to chelate calcium ions (Ca ²⁺ ), which are the major functions of cell adhesion factors. As a result, as shown in FIG. 11, no sphere was formed upon addition of EDTA. In other words, it was confirmed that the formation of mesenchymal stem cells was prevented by the addition of ED ^2, which is a Ca ²⁺ chelator, and it can be seen that the formation of the mesenchymal stem cells is caused by Ca ²⁺ dependent cell adhesion molecule.

Therefore, the changes in the specific formation process of Ca ²⁺ -dependent cell adhesion factors, N-cadherin and E-cadherin, were examined using western blot. As can be seen in FIG. 12, in the case of N-cadherin (abeam, abl8203), it appears to disappear as the spherical formation is induced by anchorage deprivation, which is known as E-cadherin.

(abeam, abl416) was found to increase its expression as sphere formation was induced. α-tubulin was used as a control.

In Western blot, cells were lysed with reducing agent [Lysis PreMix (4 ^° C stock) + NaF (10M, xlOO) + Osovanadate (orthovanadate, 200 mM, x200) + Protease Inhibitor Cocktail (1 tablet / lOml)]. After application, SDS-polyacrylamide gel was subjected to electrophoresis and PVDF transfer membrane,

Millipore) and changes in protein expression were confirmed by primary antigen-antibody reaction and secondary antigen-antibody reaction using anti-rabbit IgG and anti-mouse IgG. From the above results, it was confirmed that E-cadherin may act as a major factor in the formation of spherical cells of human mesenchymal enjoyment cells. Therefore, the present inventors have found that E-cadherin may be involved in the formation of mesenchymal stem cells and high activity of globular cell mass. The following experiment was conducted to verify the specific effect. Example 4 Confirmation of E-cadherin Activity-Effect on Spheroid Formation and High Activity of Spheroid Cell Mass (1) Effect on Spheroid Formation Effect of E-cadherin on Spheroid Formation of Mesenchymal Stem Cells I looked at it.

 Specifically, E-cadherin neutralization antibody (Clone, DECMA-1) was used, which removes the intercellular adhesion of E-cadherin by using an antibody that recognizes and attaches the cell membrane of E-cadherin. to be.

 This is a method of adding 2-10 g / ml of E-cadherin neutralization antibody or IgG when inducing formation in bFGF-free ESM medium in a low adhesion dish.

As can be seen in FIG. 13, spheres were formed in the group treated with IgG and the cell control group treated with nothing (naive), but sphere formation was not achieved in the E-cadherin inhibitory group (neu E-cad). It was. The Naive group is intended to confirm that the conditions of the antibody treatment group are not special conditions for killing or activating cells, and usually yield the same result compared to the IgG group. In addition, the effect of the adenoviral vector (E-cadherin adenoviral vector) on overexpression of E-cadherin was investigated. Revalidation. In the same vector, a vector containing LacZ gene instead of E-cadherin was used as an E-cadherin adenoviral vector control.

 The CMV promoter was used and the adeno viral vector was used after induction of viral packaging in 293 cells. Like normal viral vector transduction, viral supernatant was fixed and cultured and added to 70% confluence of mesoderm stem cells to induce E-cadherin expression. After 24 hours of transduction, 24 hours of stabilization was induced and the cells were separated into single cells, and then spherical formation was induced in low adhesion dishes using bFGF-free ESM medium and sampled after observation.

 Cells that were not treated with adenoviral vectors (naive) were used as cell controls for adenoviral vecor (E-cadherin and LacZ) treatment groups. After processing the vector for 4 hours in the cells, the effect of inducing the formation was confirmed after 5 hours and 24 hours. The Naive group is intended to confirm that the conditions of the vector treatment group are not special conditions for killing or activating cells, and usually yield the same results compared to the LacZ group.

 As can be seen in FIG. 14, when E-cadherin was overexpressed in mesenchymal stem cells (E-cad), in contrast to the inhibition of E-cadherin function, spherical formation proceeded rapidly and induction efficiency increased. .

 From the above results, it is clear that E-cadherin is a major factor regulating the formation of mesenchymal stem cells.

(2) Effects on ERK and / or AKT Activation through phosphorylation of ERKCetracellular signal-regulated kinase (ARKT) and AKT (V ^to akt murine thymoma viral oncogene homolog) is the most important factor in indicating cell activity. to be. Therefore, experiments were first conducted to confirm their activity according to specific formation. After inducing spherical formation according to (2) of Example 1, cells were sampled every 30 minutes, 1 hour, and 3 hours. Phosphorylation of ERK and AKT is usually done after various treatments Since it was confirmed within 3 hours, it was sampled at the above time to evaluate the degree of phosphorylation.

As shown in Figure 15, it can be seen that the activated AKT (pAKT) and ERK (pERK) of the total AKT (tAKT) and ERK (tERK) increases as the globular cell mass is formed by anchorage deprivation. From the above results, the formation of globular cell mass due to the floating culture of mesenchymal stem cells shows the result of activating both AKT and ERK, which also indicates that the AKT and ERK pathway may be an active pathway according to the formation of human mesenchymal stem cell spheres. This is a suggestive result. Next, an experiment was conducted to determine whether E-cadherin directly regulates these active factors. Specifically, E-cadherin neutralization antibody (clone DECMA-1, sigma) using a single cell was treated, induction of sphere formation was confirmed by western blot. In Western blot, cells were lysed with a reducing agent [Lysis PreMix (4 ^° C stock) + NaF (10M, xlOO) + orovanadate (ortho vanadate, 200 mM, x200) + protease inhibitor cocktail (1 tablet / lOml)]. SDS—polyacrylamide gel electrophoresis and transfer to PVDF transfer membrane (Millipore) followed by secondary antigen-antibody reaction using primary antigen-antibody reaction, anti-rabbit IgG and anti-mouse IgG. Change was confirmed.

Specifically, the antibody that recognizes and attaches the cell membrane portion of E-cadherin is sampled after removing the intercellular adhesion of E-cadherin, and the degree of phosphorylation is evaluated. The results are shown in FIG. 16. The naive group, which refers to a cell control group that has not been treated with antibodies, is used to confirm that the conditions of the antibody treatment group are not special conditions for killing or activating cells, and generally produce the same result compared to the IgG group.

As can be seen in Figure 16, the control group IgG treated group and naive group did not change the activated pAKT and pERK, whereas E- In the cadherin inhibitory group (neuE-cad), the activity of AKT and ERK was decreased (pAKT and pERK reduction). In addition, the results were re-validated using an adenoviral vector (E-cadherin adenoviral vector) that overexpresses E-cadherin. Like normal viral vector transduction, viral supernatant was fixed and cultured and added to 70% confluence of mesoderm stem cells to induce E-cadherin expression. After 24 hours of transduction, 24 hours of stabilization was induced and the cells were isolated into single cells, and induction and sampling were performed in low adhesion dishes using bFGF-free ESM medium. A vector containing LacZ gene instead of E-cadherin was used as the E-cadherin adenoviral vector control. Naive cells that did not have adenoviral vectors were used as cell controls for adenoviral vecor (E-cadherin and LacZ) treatment groups. The Naive group is intended to confirm that the conditions in the vector treatment group are not special conditions for killing or activating cells, and usually yield the same results compared to the LacZ group.

 As can be seen in Figure 17, E-cadherin overexpression group (E-cad) was shown to increase the activation of ERK and AKT (increase pAKT and pERK) compared to naive and LacZ group.

(3) Effects on cell growth and cell death We examined the effects of E-cadherin on the growth and death of mesenchymal stem cells.

Specifically, cell growth of the E-cadherin overexpression group, naive and LacZ group was examined by flow cytometry. Formed by incubating for 24 hours after treatment with adenoviral vector overexpressing E-cadherin to the mesenchymal stem cells suspended in culture as in Example 1 (1) as in Example 4 (1) After the spheres were separated into single cells, the cell nuclei were stained, and the cell cycle was analyzed by flow cytometry. The growth of the cell is assessed by the percentage of the S phase that labels the active cell growth during the cell cycle.

 As shown in Figure 18, compared with navie and LacZ group E-cadherin overexpression group (E-cad) was found to increase the S phase, which is the main growth stage for mesenchymal stem cell activity.

 In addition, as shown in FIG. 19, it was confirmed that apoptosis was reduced because the ratio of Ml groups in which E-cadherin overexpression (E-cad) cell death occurred decreased.

(4) Effect on the secretory capacity of VEGF

The effects of e-cadherin on the secretory capacity of VEGF (Vascular endothelial growth factor) of mesenchymal stem cells were examined. VEGF is a major factor in the improvement of ischemic heart disease.

 Specifically, ELISA using real time PCR and antigen antibody reaction was performed for the E-cadherin overexpression group, naive and LacZ group, and the mRNA and protein levels were compared, respectively.

 After 48 hours of treatment with the adenoviral vector to the cells, RNA was extracted from the cells, cDNA was synthesized, and RNA levels were quantitatively measured using VEGF specific primers (Bionian (Korea) custom-made) (VEGF-real-time). PCR), the cells were treated with an adenoviral vector and 48 hours later, the VEGF antigen-antibody reaction was induced to the cultures, and the amount of VEGF at the protein level was measured (VEGF—ELISA).

 As can be seen in FIG. 20, VEGF was shown to be increased in both E-cadherin overexpression (E-cad) mRNA and protein levels.

These results show that E-cadherin is not only an induction factor for the formation of human mesenchymal stem cells, but also acts as a regulator of various in vivo activities. In conclusion, it is clear that E-cadherin promotes the spheroid formation of human mesenchymal stem cells and further induces high activity of globular cell mass. Example 5: Test to improve the immunomodulatory ability of cord blood-derived mesenchymal stem cells by aggregate formation

(1) To verify the effect on the immunomodulatory ability of cord blood-derived mesenchymal stem cells after MLR (Mixed Lymphocyte Reaction) formation, the donor was tested for immunomodulatory activity of two different cord blood-derived mesenchymal stem cells in vitro. It was evaluated through the MLR (Mixed Lymphocyte Reaction) method.

 Specifically, two different human-derived homologous human peripheral blood cells were co-cultured to induce an autoimmune reaction, and then, each cell culture was inhibited, followed by plate-cultured cord blood-derived mesenchymal stem cells (monolayer stem cells). ) Or incubated with umbilical cord blood-derived mesenchymal stem cells (aggregate stem cells).

Umbilical cord blood-derived mesenchymal stem cells were cultured in a monolayer in ΜΕΜ-α medium contained in 10% FBS 7} in a 175T culture dish at a rate of 5 X 10 ⁵ / cm ² and used for experiments after 80-90% growth. Umbilical cord blood-derived mesenchymal stem cells were treated with 10 jag / ml mitomycin C for 1 hour in suspension and then used for plate attachment and aggregate formation.

To form aggregates, cord blood derived mesenchymal stem cells treated with mitomycin C were treated with DMEM / F12 (20% Knock out SR, O.lmM β-mercaptoethane, 1% non-essential amino acid, 50 IU / ml penicillin, 50 ug / ml streptomycin) medium was incubated for 24 hours by hanging drop method on the culture dish lid under 2 X 10 ³ cells / 20uL medium conditions.

^Four single-layered stem cells 2X10 and ^three stem cells 2X10 ³ were put in a well of a 96-well plate and used as a negative control. Positive control Wells were co-cultured with two different human peripheral blood cells of 2xl0 ⁵ to cause allogeneic reaction. And in the test group well was confirmed that the key when suppressing monolayer stem cells 2X10 ⁴ gae eu aggregates stem cells 2X10 ³ gae to one another ssaeyang into ⁵ each 2xl0 the two other human peripheral blood cells Co to each insert well alloimmunization banung . After incubation for 5 days, the growth and colonization of the cells were confirmed under a microscope. Next, BrdU was treated on the 5th day of culture to measure DNA of newly synthesized cells for the last 24 hours.

 As a result, as shown in FIG. 21, since the stem stem cell (s) suppresses more than 37% of allogeneic immune reactions compared with the tomography stem cell (M), it was confirmed that the stem stem cell has an excellent ability to suppress the immune response. It was.

(2) Confirmation of PGE ₂ (prostaglandin E2) secretion level

The PGE ₂ secretion level known as an immunomodulator in the MLR culture obtained in (1) was measured using an ELISA method (Cayman Chemical Company, prostaglandin E2 ELISA Kit (catalog No. 514010)). The experimental culture was reacted with capture antibody at 4 ^° C. for 18 hours, and the reaction was allowed to react for 90 minutes at room temperature. As a result of ELISA analysis, it can be seen that PGE ₂ secretion is greatly increased after formation of the colon in the environment in which allogeneic immune reaction occurred as shown in FIG. 22 (N: monolayer stem cell; A: cumulative stem cell). This result means that the immunomodulatory ability of cord blood-derived mesenchymal stem cells is improved by platelet formation compared to plated stem cells.

(3) Confirmation of immunogenicity To analyze the effect of aggregate formation on the immunogenicity of umbilical cord blood-derived mesenchymal stem cells, tissue analysis was performed. Specifically, umbilical cord blood-derived mesenchymal stem cells and platelet-derived mesenchymal stem cells after cochlear formation were injected into the myocardium of an ischemic cardiovascular disease rat model, and then immune cells penetrated around the ischemic tissues of the heart. To analyze the response, cardiac tissue samples were stained with F4 / 80, an immune cell marker, and green with secondary antibody. Markers were used. The injected cells were stained with Dil prior to injection to distinguish them from other cells.

 As can be seen in Figure 23, a significantly smaller number of immune cells compared to flat blood-attached umbilical cord-derived mesenchymal stem cells (NaiVe) in the tissue (Spheroid) injecting umbilical cord blood-derived mesenchymal stem cell mass after formation of the globules Was observed. This means that the formation of glomeruli can lower the immunogenicity of cord blood-derived mesenchymal stem cells. Example 7: verification of enhanced chondrocyte death and cartilage regeneration of cord blood-derived mesenchymal stem cells by aggregate formation

(1) Inhibitory effect of chondrocyte death Since mesenchymal stem cells derived from umbilical cord blood are well known to differentiate into chondrocytes, inhibition of cell death and anti-inflammatory function by various secretion factors have been confirmed. Has been. Therefore, it was verified whether the therapeutic effect of umbilical cord blood-derived mesenchymal stem cells on cartilage cell death and cartilage regeneration due to cartilage damage and arthritis can be improved or improved through the formation of a globule.

3 ml of rabbit chondrocytes were placed in a 10 cm ² petri dish at a rate of 5 x 10 ⁴ / cm ² .

Single layer culture with DMEM (10 FBS, 50 ug / ml Gentamicin) medium and plate-cultured cord blood-derived mesenchymal stem cells (naive hUCB—MSC) at 5 x 10 ⁵ / 3ml Incubated in the. Cord blood-derived mesenchymal stem cells (MSC-spheroid hUCB) of ungjip body before co-culture 1 days, 1 x 10 ⁴ / 20uL conditions in DMEM / F12 (20% Knock out SR, O. lmM β- Murray captopril ethanol, 1 % Non-essential amino acids, 50 IU / ml penicillin, 50 ug / ml streptomycin) medium was cultured by hanging drop method on the lid of the culture dish. Six days after culturing rabbit chondrocytes, isolated co-culture was performed using a trans-well. naive hUCB—MSC co-cultures the trans-well in culture on a chondrocyte culture plate of rabbits in culture, and spheroid hUCB-MSC 50 spheroids formed were transferred to 3 ml of DMEM (10% FBS, 50 ug / ml Gentamicin) medium and co-cultured in trans-wells. At this time, 500 μΜ of sodium nitroprusside was treated under the condition of chondrocyte death.

As a result, as shown in FIG. 24A, it was confirmed that the death of chondrocytes was greatly reduced by the survival / kill staining. As shown in FIG. 24B, the degree of inhibition of chondrocyte death during spheroid formation was hUCB-MSC (1). ) Was 90.6 土 4.4% and hUCB-MSC (2) was 95.7 士 1.2%, which was significantly increased compared to the control (66.2 士 13.0%). (2) Cartilage Regeneration Effect A 10-week-old New Zealand white rabbit incision cuts the outer skin, subcutaneous and articular capsules and exposes the joints. After completion, hemostasis was performed on the damaged area using sterile gauze for 20 seconds. Then, two cell groups (High cell and Low cell group) classified according to the cartilage differentiation ability and regenerative ability of cord blood-derived mesenchymal stem cells by donor, normal human lung fibroblast cells as a control group, and Low cell The 5X10 ⁶ cells were mixed with 4% hyaluronic acid and injected into the damaged area. Subsequently, the sutures of the articular capsule, subcutaneous tissue and skin were sutured and bred for 10 weeks.

The cartilage damage scores obtained through visual analysis and tissue staining analysis of cartilage injuries after 10 weeks of FIG. 25 (the lower the damages were restored by cartilage regeneration), Pineda et al. (1992, Acta Anat (Basel)) and Wakitani et al. According to (1994, J Bone Joint Surg Am) method, the high cell with less repair of cartilage damage (5.002 2.24) and the low cell (6.00 土 1.22) formed with agglomerates were high, and the low cell (7.40 土 1.52) was evaluated. And control cells (7.20 土 1.48) were as low as expected. Therefore, these results imply that the formation of the globules enhances the efficacy of umbilical cord blood-derived mesenchymal stem cells on cartilage cell death and cartilage regeneration. Example 8 Verification of Enhancement of Lung Regeneration Effect in Lung Injury Model of Umbilical Cord Blood-derived Mesenchymal Stem Cells by Globule Formation (1) Inhibition of Cell Death of VEGF-expressing Umbilical Cord Blood-derived Mesenchymal Stem Cells Related to Lung Cell Regeneration Inflammation and the like are caused by various secretion factors. In particular, VEGF is known in relation to pulmonary capillary regeneration function, lung cell proliferation and suppression of lung cell death in lung injury disease [Varet J. et al., J Physiol Lung Cell Mol Physiol, 298, L768-L774 ( 2010); Kuhn H et al., Respirology 15, 343-348 (2010)]. Increased VEGF secretion of umbilical cord blood-derived mesenchymal stem cells after formation of the glomerulus can be expected to affect lung regeneration in lung injury models. ELISA was performed using VEGF antibody (R & D systems, ELISA kit cat # DY293B). As a result, it was confirmed that the secretion of VEGF increased more than three times after formation of the globules. This means that it may be an important case in improving recovery of lung injury due to increased secretion of VEGF, which can be an important therapeutic factor in lung injury.

(2) Enhancement of Differentiation of Umbilical Cord Blood-derived Mesenchymal Stem Cells into Pulmonary Cells by Globule Formation

Umbilical cord blood-derived mesenchymal stem cells are well known for their differentiation into pulmonary cells, and they have been engrafted into lung tissue to produce or regenerate pulmonary cells, suppress pulmonary fibrosis symptoms, and anti-inflammatory functions. . In particular, SP-C (surfactant protein C) protein secreted by lung cells for lung regeneration in lung injury disease is important. Therefore, cord blood for lung regeneration It was verified whether the therapeutic effect of the derived mesenchymal stem cells can be improved or improved through the globules.

In order to verify the effect of umbilical differentiation of cord blood-derived mesenchymal stem cells after aggregate formation, donors were evaluated for lung differentiation of two different cord blood-derived mesenchymal stem cells through in vitro SP-C gene expression. . Umbilical cord blood-derived mesenchymal stem cells were grown to 50-60% by monolayer culture in ΜΕΜ-α medium containing 10% FBS in a 175T culture dish at a 5 X 10 ³ / cm ² ratio. Umbilical cord blood-derived mesenchymal stem cells cultured in a monolayer are cultured in a 75Τ culture dish.

Aggregation was performed by comparing the hanging drop method and the bioreactor method. Umbilical cord blood-derived mesenchymal stem cells cultured in hanging drop cells were treated with DMEM / F12 (20% Knock out SR, O.lmM β— mercaptoethanol, 1% Non-essential amino acid, 50 IU) under 5 X 10 ⁴ / 20μb conditions. / ml penicillin, 50 ug / ml Streptomycin) medium was incubated for 24 hours in a petri dish lid. After checking the proliferation and spheroid formation of the reaction cells under a microscope, it was transferred to a 35 pie culture dish and cultured. Umbilical cord blood-derived mesenchymal stem cells cultured with bioreactor aggregates were cultured at a constant speed of 70 rpm in a spinner flask at 5 X 10 ⁵ / ml.

 After culturing for 5 days, the cell group cultured in monolayer and the cell population cultured in a globule were extracted with RNA and synthesized cDNA. Next, SP-C gene expression levels were analyzed by real time PCR.

As shown in FIG. 26, SP-C expression of cord blood-derived mesenchymal stem cells after hanging drop colony formation was significantly improved compared to the general plate attachment culture and bioreactor aggregate culture. This means that the therapeutic efficacy may be more desirable compared to the bioreactor population. Using the Hollow Object by the Hanging Drop Method It was confirmed that the expression of pulmonary differentiation factor SP-C was increased by 15-80 times compared with the bio-actor aggregates and 2--8 times higher than that of single cells. This means that it may be an important case in improving the recovery of lung injury by increasing the expression of SP-C, which can be an important therapeutic factor in lung injury. From the results of the above embodiment, the cord blood-derived mesenchymal stem cells improve the treatment efficacy in the damage and inflammatory diseases such as cartilage and lung as a cell therapy due to the increase of various functional secretion factors and the decrease in immunogenicity from the formation of the globules. It is expected to be used as an expected technology. Example 9 Method of Forming Globules of Cord Blood-derived Mesenchymal Stem Cells Using a Rocker

Rock formation of the MSCs was used to lock the MSCs to induce the formation of the aggregates. 10,000-20,000 cells / cm ² of MSC cells were used, and the medium composition was alpha-DMEM medium containing 10% FBS. Culture conditions were placed compact rocker CR95 (FinePCR) in 37 ^° C. and C0 ₂ incubator, and incubated for 24 hours at a locking speed of 8-12 rpm. A non-treated bacterial culture dish was used to prevent MSC adhesion of the dish surface.

 As a result, an increase in locking speed caused a decrease in MSC sparsity. In addition, the size of the aggregate produced was produced differently according to the locking speed, it can be seen that the size varies within one locking speed. Although the present invention has been described in connection with the specific embodiments above, those skilled in the art may variously modify and change the present invention within the scope of the present invention as defined by the appended claims.

Claims

Claims 1. A method of producing a highly active human mesenchymal stem cell mass, comprising culturing the human mesenchymal stem cell against force and forming a globular cell mass. 2. The method of claim 1, wherein the culturing is culturing the mesenchymal stem cells in a drop of medium opposed to gravity. 3. The method of paragraph 1, wherein the culturing is performed using a serum excretion medium comprising serum replacement (SR). 4. The method of paragraph 3, wherein the serum excrement medium is a human embryonic stem cell culture medium that does not contain a basic fibroblast growth factor (bFGF). 5. The method of claim 1, wherein the mesenchymal stem cells are human umbilical cord blood-derived mesenchymal stem cells. 6. The method of producing highly active human mesenchymal stem cell mass, comprising culturing human mesenchymal stem cells to form globular cell mass, and increasing the amount of E-cadherin in the mesenchymal stem cell during the culture. . 7. The method of claim 6, wherein the increase in the amount of E-cadherin is performed by introducing an expression vector of E-cadherin into the mesenchymal stem cells. 8. Highly active human mesenchymal stem cell mass produced by the method of any one of items 1-7. 9. A cell therapy comprising the highly active human mesenchymal stem cell mass of claim 8. 10. The cell therapeutic agent according to 9, wherein the cell therapeutic agent is used for formation of adipocytes, bone cells, chondrocytes, muscle cells, nerve cells, cardiomyocytes, hepatocytes, pancreatic beta cells, vascular cells or lung cells. Cell therapy. 11. The method of claim 9, wherein the cell therapy is used to treat lung disease; Suppression or treatment of inflammation caused by lung disease; Lung tissue regeneration; And pulmonary tissue fibrosis suppression, wherein the cell therapy agent is used for any one selected from the group consisting of. 12. The cell therapy according to item 9, wherein the cell therapy is used for the treatment of cardiovascular disease. 13. The cell therapy according to item 9, wherein the cell therapy is used to treat angiogenesis. 14. The cell therapy according to item 9, wherein the cell therapy increases the immunomodulatory function. 15. The cell therapy according to item 9, wherein the cell therapy lowers any one of immunogenicity, immune cell infiltration, or immunogenicity. 16. The cell therapy according to item 9, wherein the cell therapy is for cartilage regeneration. 17. The cell therapy according to item 9, wherein the cell therapy inhibits inflammation. 18. A cell therapy method comprising administering the highly active human mesenchymal stem cell mass of paragraph 8 to a subject in need thereof. 19. The method of paragraph 17, wherein the cell therapy method comprises the formation of adipocytes, bone cells, chondrocytes, muscle cells, neurons, cardiomyocytes, hepatocytes, pancreatic beta cells, vascular cells or lung cells; Lung disease treatment; Suppression or treatment of inflammation caused by lung disease; Lung tissue regeneration; Suppression of pulmonary tissue fibrosis; Treatment of cardiovascular disease; Increase in immunomodulatory function; Degradation of any of immunogenicity, immune cell infiltration or immunogenicity; Cartilage regeneration; Or a cell therapy method used for inhibiting an inflammatory response.