KR20110115688A - Method for increasing stem cell activity and stem cells produced by thereof - Google Patents

Abstract

The present invention relates to a method for increasing the activity of human stem cells, specifically characterized in that it comprises the step of treating endothelin-1 (Endothelin-1) in culture of human stem cells. In another aspect, the present invention provides a highly active human stem cell prepared by the above method, a cell therapeutic agent comprising the highly active human stem cell. According to the present invention, it is possible to increase the activity of human mesoderm stem cells of various origins, and ultimately, it is expected to be very useful for developing high-efficiency cell therapeutics.

Description

Method for increasing stem cell activity and stem cells produced by the method

The present invention relates to a method for increasing the activity of human stem cells, highly active stem cells produced by the method, and a cell therapeutic agent comprising the highly active stem cells.

Stem cells are cells capable of differentiating into the 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 cell division is stopped due to differentiation, proliferation is possible due to cell division (self-renewal). It can be differentiated into other cells by different environment or differentiation stimulus, which is characterized by having plasticity in differentiation.

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

Embryonic stem cells are formed from the inner 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 in the undifferentiated state without being killed. Unlike adult stem cells, germ cells can be produced, and thus they can be inherited (Thomson et al., Science, 282: 1145-1147, 1998; Reubinoff et al.). , Nat. Biotechnol., 18: 399-404, 2000).

Human embryonic stem cells are prepared by separating and culturing only inner cell mass when forming human embryos. Currently, human embryonic stem cells made worldwide are obtained from frozen embryos remaining after infertility. Various attempts have been made to use human embryonic stem cells, which are capable of differentiating into all cells, as cell therapy, but have not yet fully controlled the risk of cancer and the high wall of immune rejection.

Recently, as a complementary measure, induced pluripotent stem cells (iPS) have been reported. IPS, which is included in the concept of pluripotent stem cells, is a cell which has redifferentiated differentiated adult cells in various ways and returned to embryonic stem cells, which are the early stages 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, autologous cells can be used to exclude the risk of immune rejection, 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. Mesoderm stem cells are pluripotent cells capable of differentiation into adipocytes, bone cells, chondrocytes, muscle cells, neurons, cardiomyocytes, etc. and are known to have a function of regulating immune responses.

Mesoderm 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 swirling shape, and express the basic cell surface markers CD73 (+), CD105 (+), CD34 (-), and CD45 (-). It is generally defined as mesodermal stem cells.

In this regard, for mesodermal stem cells having different genetic origins and / or backgrounds, there is usually no significant difference from each other with respect to the criteria for defining the existing mesodermal stem cells mentioned above, but usually their respective in vivo activity. In terms of the difference. In addition, when mesodermal stem cells are used as a cell-derived cell therapy, the available pool is limited, so even if the in vivo activity of the mesodermal stem cells is low, there is no choice and replacement is impossible.

In addition, mesenchymal stem cells are generally required to meet the minimum number of cells required for regenerative medicine and / or cell therapy (approximately 1 × ^{10 9} ) in order to be used as cell therapy. If so, the number of cells needed is increased. Therefore, in order to supply this amount from existing mesoderm stem cells of various origins, at least 10 passages are required in vitro (in vitro). In this case, the cells are aged and deformed, and thus no longer serve as cell therapeutics. The problem arises that is not suitable to achieve.

In view of the above problems, finding a way to maximize the activity of mesoderm stem cells and reduce the number of applied cells using efficiency-enhanced stem cells is a task of the art for the practical application of regenerative medicine and cell therapy. It can be said.

The present invention has been made by the above-described needs, by increasing the activity of human mesoderm stem cells and thereby increasing the efficiency of stem cells required in the field of regenerative medicine and cell therapy, human mesoderm stem cells of various conventional sources In order to overcome the limitation of the efficiency of the mesoderm stem cells to increase the practicality as a therapeutic agent.

In order to solve the above problems, the present invention provides a method for increasing the activity of human mesoderm stem cells. Specifically, the present invention is characterized in that it comprises the step of treating the endothelin-1 (Endothelin-1, ET-1) in the culture medium of mesoderm stem cells. In another aspect, the present invention provides a highly active human stem cell prepared by the above method, a cell therapeutic agent comprising the highly active stem cell.

The present invention can be used as a method for maximizing the in vivo efficiency of human mesoderm stem cells, which is the best resource of cell therapy, and the present invention can be applied to both human mesoderm stem cells of various origins and various culture conditions currently used. Provide a broad range of defined activity enhancement methods. Ultimately, the present invention is expected to contribute to the advancement of the practical use of cell therapy by increasing the efficiency of human mesoderm stem cells as a cell therapy, and further contribute to the development of treatment for intractable diseases such as cardiovascular diseases and neurological diseases. It is expected.

Figure 1 shows the effect of endothelin-1 on the growth of mesoderm stem cells.
Figure 2 shows the effect of endothelin-1 on the change in the cell cycle of mesoderm stem cells.
Figure 3 shows the effect of endothelin-1 (change in VEGF RNA level) on the secretory capacity of mesoderm stem cells.
Figure 4 shows the effect of the endothelin-1 (change in VEGF Protein level) on the secretory capacity of mesoderm stem cells.
Figure 5 shows that the endothelin receptor A is specifically expressed in mesoderm stem cells.
Figure 6 shows the expression changes of VEGF after BQ123 treatment is an endothelin receptor A specific blocker (blocker).
Figure 7 shows the results of comparing the size of the heart, when injected into the ischemic cardiovascular rat model mesenchymal stem cells treated with endothelin -1.
Figure 8 shows the results of quantifying the electrocardiogram when the endoderm-1 treated mesoderm stem cells were injected into an ischemic cardiovascular rat model.

The present invention relates to a method for increasing the activity of human mesoderm stem cells having various genetic backgrounds and / or origins, characterized in that it comprises the step of adding endothelin-1 to the mesoderm stem cell culture medium.

The inventors performed DNA arrays on human mesoderm stem cells having different in vivo activity, and endothelin was studied as a result of intensive studies on genes showing relatively high expression in cells showing the greatest in vivo activity. The present invention was completed based on the fact that -1 increases the activity of the mesoderm stem cells.

Endothelin-1 is a peptide consisting of 21 amino acids mainly produced in vascular endothelial cells. The first occurrence after gene transcription is prepro ET-1, which consists of 212 amino acids, and the enzyme produces big ET-1, which consists of 38 amino acids. Big ET-1 is the end product of endothelin-1 produced by the end of the C-terminal amino acid by the action of the endothelial converting enzyme (ECE). Endothelin-1 has attracted the attention of many researchers because of its strong and persistent vasoconstriction. Endothelin-1 is also produced in vascular smooth muscle cells, but major biosynthesis occurs in vascular endothelial cells.

The production of endothelin-1 is promoted by thrombin, angiotensin II, vasopressin, transforming growth factor β, tumor necrosis factor α, hypoxia and oxidized LDL and is inhibited by endothelium-derived relaxing factor (EDRF, nitric oxide). In addition, shear stress, a physiological stimulus, which pushes the walls of blood vessels in the direction of blood flow, promotes the production of endothelin-1 when the stimulus is weak, but suppresses it when it is large.

Ischemic heart disease is a disease caused by myocardial dysfunction or necrosis due to stenosis or obstruction of coronary arteries. There are various findings suggesting that endothelin-1 may be involved in pathophysiology. First, the increase in blood endothelin-1 in the acute phase of myocardial infarction patients. Cardiomyocytes of cardiac cells undergoing ischemia-reperfusion are known to increase the number of endothelin receptors, promote the production and secretion of endothelin-1 from the myocardium, and increase the contractile responsiveness to endothelin-1 of coronary arteries. These findings suggest that endothelin-1 may play a pathophysiological role in myocardial infarction. Under the assumption that endothelin-1 will affect the size of myocardial infarction, there have been a number of studies that observed that endothelin antagonists could reduce the size of infarcts in animals. However, the results are difficult to draw clear conclusions with the report that there is no antagonist effect and no report.

Considering the action of endothelin-1 considered in the above, the present invention reversely treated endothelin to human mesoderm stem cells to increase the activity and then to see the effect on ischemic myocardial infarction. Therefore, the present invention is intended to increase the activity of mesodermal stem cells to increase the efficiency as a cell therapy, and to find new applications and indications of endothelin-1.

The present invention provides a method for increasing the activity of mesoderm stem cells, comprising the step of pretreating endothelin-1 to human mesoderm stem cell culture medium.

Human mesoderm stem cells used in the present invention are not limited in their genetic background and / or origin. For example, human cord blood-derived mesoderm stem cells can be used.

The present inventors examined the effects of endothelin-1 on mesodermal stem cell activity after pretreatment with endothelin-1 in human mesoderm stem cell culture. First, as a result of examining the effect on the growth of mesoderm stem cells, it was confirmed that the mesenchymal stem cells pretreated with endothelin-1 increased in cell number compared to the control group without pretreatment with endothelin-1 (see FIG. 1). In addition, as a result of examining the effect on the change of the cell cycle, it was confirmed that the S group (synthetic group) is increased (see Fig. 2).

From the above results, the present invention means that the mesenchymal stem cells can be a method of preventing aging in vitro culture and at the same time increasing the number of cells. In other words, pretreatment with endothelin-1 can delay aging as well as delay the aging of mesenchymal stem cells to obtain the number of cells required for regenerative medicine and cell therapy. Yields of mesenchymal stem cells will be obtained.

In addition, the effects of endothelin-1 on the mesenchymal stem cell secretion ability, VEGF (vascular endothelial growth factor, a major paracrine factor in improving cardiovascular disease) endothelin- 1 pretreatment was confirmed to increase both at the RNA and protein levels (see Figures 3 and 4). In order to verify that the increase in VEGF is caused by endothelin-1, it was first confirmed that A of endothelin receptors A and B is specifically expressed in mesodermal stem cells (see FIG. 5), and a receptor A specific blocker (see FIG. 5). ) BQ 123 was treated to observe the expression changes of VEGF. A decrease in RNA and protein levels of VEGF after the blocker treatment was confirmed (see FIG. 6), and from the results it was evident that the increase in RNA and protein levels of VEGF was due to endothelin-1.

Next, the effects of endothelin-1 on the in vivo activity of mesoderm stem cells were examined. Ischemic cardiovascular rat models can be used to assess in vivo activity. Ischemic cardiovascular rat model refers to an animal ischemic cardiovascular disease model created by inducing ischemic state by ligation of coronary artery of heart. The present inventors injected mesenchymal stem cells cultured by pretreatment with endothelin-1 to the peritoneal cardiac muscle of the model and observed the results. First, as a result of comparing the size of the heart, it was observed that the endothelin-1 treatment group was significantly smaller in size than the control group (see FIG. 7), which is due to fibrosis due to the death of cardiomyocytes in ischemic heart disease. Means that it has an effect of significantly reducing the increase of.

In addition, four weeks after injecting mesodermal stem cells into the ischemic model, the electrocardiogram may be measured to evaluate the in vivo activity. Specifically, Left ventricular end-diastolic dimension (LVEDD), Left ventricular end- systolic dimension (LVESD), Left ventricular end-Fractional Shortening (LVFSD), Left ventricular bleeding rate (Left ventricular end-Ejection Fraction (LVEF)) can be quantified to compare the improvement. The left ventricular fractional shortening (LVFS) is defined as LVEDD-LVESD / LVEDD, and the left ventricular blood glucose (LVEF) is defined as LVEDD ² -LVESD ² / LVEDD ² . The smaller the left ventricular diastolic diameter (LVEDD) and the left ventricular systolic diameter (LVEDD), the higher the left ventricular fractional shortening (LVFS) and the left ventricular volume (LVEF). The left ventricular fractional shortening (LVFS) and the left ventricular ejection fraction (LVEF), which are markers of improvement, were significantly higher in the endothelin-1 treated group than in the control group (see FIG. 8).

From the above results, it is clear that endothelin-1 enhances the activity of human mesoderm stem cells, and thus the present invention can be used as a method for maximizing the in vivo activity of human mesoderm stem cells of various origins.

Therefore, the present invention provides a method for producing highly active human mesoderm stem cells, the method comprising: attaching and culturing the mesoderm stem cells using a human mesoderm stem cell culture medium; And adding endothelin-1 to the culture medium. In addition, the present invention provides a highly active mesoderm stem cells with increased activity by the above method. When using the highly active stem cells in the field of regenerative medicine and cell therapy, the same effect can be expected with a small number of cells compared to the conventional stem cells. In another aspect, the present invention provides a cell therapy containing the highly active stem cells.

In addition, the present invention provides a kit for preparing a highly active mesoderm stem cell, which kit may include α-MEM, FBS, and endothelin-1. Use of the kit may be a prescribed method of enhancing the activity of human mesoderm stem cells of various conventional origins and / or various culture conditions.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but 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 umbilical cord blood-derived mesodermal stem cells provided by MEDIPOST Co., Ltd. were used. The cells were selected by performing human mesoderm stem cell identification experiments, and expressed more than 95% of positive cell markers (CD29, CD44, CD73, CD105, CD166, HLA-ABC) and negative cell markers (CD34, CD45) of mesoderm stem cells. , HLA-DR) was identified and classified as "human umbilical cord-derived mesoderm stem cells" after confirming uniform expression of less than 5% and confirming the multiple differentiation capacity of mesoderm stem cells.

Example  1. Of Human Mesoderm Stem Cells In vitro ( in in vitro Activity evaluation

In vitro activity was evaluated using the mesoderm stem cell group pre-treated with endothelin-1 and the control group without endothelin-1 pretreatment. It will be described in detail below.

(1) Effect on growth

The hMSCs were cultured attached using α-MEM (Minimum Essential Medium, Invitrogen Co.) + 10% FBS (fetal bovine serum, bovine serum) culture medium ^{(5X10 5/60 @ dish)} . After 24 hours of culture, 10 nM of endothelin-1 was added to the medium and incubated for 72 hours.

Thereafter, trypsin-treated cells were isolated into single cells and counted in a hematocytometer. At this time, the same cell group not treated with endothelin-1 was used as a control.

As shown in Figure 1, it can be seen that the cell proliferation occurred actively in the endothelin-1 treatment group compared to the control group.

(2) effect on cell cycle changes

The mesoderm stem cells were attached and cultured using α-MEM + 10% FBS medium ( ⁵ × ¹⁰ 5/60 @ dish). After 24 hours of culture, 10 nM of endothelin-1 was added to the medium and incubated for 72 hours.

Thereafter, the cells were separated by trypsin treatment and then separated into single cells, and then the cell cycle was confirmed by flow cytometry (FACS) using PI staining nuclei. At this time, the same cell group not treated with endothelin-1 was used as a control.

As can be seen in FIG. 2, the synthesizer S group, which is a marker of cell activity and proliferation, was increased by about two times by the endothelin-1 treatment, while the resting G0-G1 group was decreased.

(3) On secretory capacity  Effect

The effects of endothelin-1 on the secretion of VEGF, a major paracrine component, in the improvement of cardiovascular disease were investigated. First, real-time PCR was performed to see the change in RNA level. Specifically, VEGF-specific primers were prepared and RNA was extracted 1, 6, 24, and 72 hours after endothelin-1 treatment to synthesize complementary cDNA, and real-time polymerization was performed using the template as a template. As shown in Figure 3, from 1 hour after the endothelin-1 treatment, the amount of VEGF RNA significantly increased compared to the control.

In addition, experiments were conducted to confirm changes in protein levels of VEGF. Specifically, the amount of VEGF secretion was confirmed by ELISA using antibody antigen reaction after harvesting the culture medium of the RNA experimental group. As shown in Figure 4, it can be seen that the secretion amount of VEGF is significantly increased in the endothelin-1 treatment group, and it is confirmed that the difference with the control group becomes larger over time.

From the above results, it can be seen that the VEGF RNA increased by the endothelin-1 treatment is synthesized into the VEGF protein and is well maintained stably.

In addition, experiments were conducted to verify that the increase in VEGF is due to the progression of the endothelin-1 binding to the endothelin receptor.

First, endothelin receptor (Receptor) A and B expression in human mesoderm stem cells was confirmed. Specifically, the endothelin receptor A, B specific primers were prepared, RNA was extracted to synthesize complementary cDNA, and the polymerization was performed using the template as shown in FIG. 5. GAPDH was used as a control. As can be seen in Figure 5, it was confirmed that among the endothelin receptor (Receptor) A and B, the receptor A is expressed in mesodermal stem cells.

According to the above results, the expression changes in RNA and protein levels of VEGF were observed upon blocker treatment of endothelin receptor A. Specifically, endothelin using BQ123 (cyclo (da-aspartyl-l-propyl-d-valyl-l-leucyl-d-tryptophyl, manufactured by Sigma) commonly used among the blockers of endothelin receptor A BQ123 10uM was first treated before -1 treatment, and after 30 minutes, endothelin-1 10nM was added to the culture medium for 24 hours, followed by changes in RNA and protein levels of VEGF. The group to which only tellin-1 was added was used as a control, and the result is shown in FIG. 6.

As can be seen in FIG. 6, RNA and protein levels of VEGF that were increased by endothelin-1 were shown to be reduced by BQ123, an endothelin receptor A blocker.

In conclusion, RNA and protein levels of VEGF were increased due to the progression of endothelin-1 to the endothelin receptor, indicating that endothelin-1 is also involved in the regulation of secretory capacity of mesodermal stem cells. .

Example  2. Of Human Mesoderm Stem Cells In vivo ( in vivo Activity evaluation

In vivo activity was evaluated using mesoderm stem cells cultured by pretreatment with the endothelin-1 and a control group without pretreatment.

An ischemic cardiovascular rat model was used to assess the in vivo activity. The ischemic cardiovascular rat model refers to a coronary artery of the heart. Refers to an animal ischemic cardiovascular disease model made by ligation to induce ischemic state.

⁵ x rats of mesoderm stem cells pre-treated with endothelin-1 were cultured and injected into the disease model animals, and 4 weeks after the injection, echocardiography was performed to analyze the improvement of the disease. After echocardiography, rats were sacrificed and heart tissue sampled and compared. At this time, after induction of ischemic injury, PBS injection group was used as a control after induction of ischemic injury to the mesoderm stem cell injection group (control group (endothelin-1 untreated cell injection group) and endothelin-1 treatment cell injection group). Only the ischemic induction group without PBS injection was used as a control for the cells or PBS injection group. At least 5 rats were used for each group above.

As can be seen in FIG. 7 showing the result of comparing the heart size, the endothelin-1 treated group (ischemic heart + endothelin-1 treated cells) was untreated control (ischemic heart + endothelin-1 ratio). The size of the heart was observed to be significantly smaller than that of the treated cells, indicating a size similar to that of the normal heart. Since the size of the heart is increased due to the death of cardiomyocytes during ischemic heart disease (see ischemic induction heart of FIG. 7), the relatively small size of the heart means that the heart disease is improved.

In addition, as shown in FIG. 8 showing the results of quantifying echocardiography, left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were markedly high in the endothelin-1 treated group. This is generally due to the fact that, when the heart is exposed to ischemia, fibrosis of the heart wall causes thinning of the heart wall, loss of motility and volumetric expansion, which leads to a decrease in LVF and LVEF. Injecting the endothelin-1 treated cells, despite exposure to ischemia, significantly reduced symptoms such as thinning of the heart wall, loss of motility and volume expansion, such as normal progression.

From the above results, it is clear that when the mesoderm stem cells pretreated with endothelin-1 are used as cell therapeutic agents, an excellent improvement effect in cardiac disease can be expected as compared with the mesenchymal stem cells without endothelin-1 pretreatment.

In conclusion, the present invention can be used as a method for maximizing the in vivo efficiency of human mesoderm stem cells, which is the best resource of cell therapy, and the present invention can be applied to both human mesoderm stem cells of various origins and various culture conditions currently used. It provides a wide range of defined activity enhancement methods that can be applied. Ultimately, by enabling the use of highly active mesodermal stem cells with increased activity by the method of the present invention, it may contribute to increasing the efficiency of human mesodermal stem cells as a cell therapy and further promoting the practical use of cell therapy.

The above description is merely illustrative of the technical idea of the present invention, and various modifications, changes, and substitutions may be made by those skilled in the art without departing from the essential characteristics of the present invention. will be. Therefore, the embodiments disclosed in the present invention and the accompanying drawings are not intended to limit the technical spirit of the present invention, but are intended to be described, and the scope of the technical idea of the present invention is limited by the embodiments and the accompanying drawings. It is apparent to those skilled in the art that no.

Claims (7)

A method of producing highly active human mesoderm stem cells comprising the following steps:
a) attaching and culturing human mesoderm stem cells using human mesoderm stem cell culture medium; And
b) adding endothelin-1 to the culture medium. The method of claim 1, wherein the culture medium is α-MEM (Minimum Essential Medium) + 10% Fetal Bovine Serum (FBS) medium. According to claim 1, wherein the endothelin-1 (Endothelin-1) is characterized in that the addition of the mesoderm stem cells after the attachment culture 24 hours. The method of claim 1, wherein the human mesoderm stem cells are human umbilical cord blood-derived mesoderm stem cells. Highly active human mesoderm stem cells produced by the method of any one of claims 1 to 4. Cell therapy comprising the highly active human mesoderm stem cells of claim 5. Highly active human mesoderm stem cell preparation kit comprising:
a) Minimum Essential Medium (α-MEM);
b) FBS (fetal bovine serum, bovine serum); And
c) Endothelin-1.

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