阅读说明：本技术 间充质干细胞的制造方法、间充质干细胞的治疗效果标志物和治疗效果判断方法、以及包含间充质干细胞的细胞制剂 (Method for producing mesenchymal stem cell, marker for treatment effect of mesenchymal stem cell, method for determining treatment effect, and cell preparation containing mesenchymal stem cell ) 是由 千见寺贵子 藤宫峰子 斋藤悠城 中野正子 大谷美穗 水江由佳 松村崇史 神谷梢 于 2018-04-25 设计创作，主要内容包括：本发明的目的在于,提供包含治疗效果好的MSC的细胞制剂。提供一种活化的MSC的制造方法,包括使用具有由纤维构成的三维结构的细胞培养载体在含有哺乳动物胎儿附属物来源的活化剂的培养基中培养MSC的工序。此外,还提供选自由p16<Sup>ink4a</Sup>、p14<Sup>ARF</Sup>、CDK4、CDK6、RB和CD47组成的组的MSC治疗效果标志物、使用同一标志物的治疗效果判断方法、判断MSC对用于提高治疗效果的处理的适应性的方法、包含MSC的细胞制剂及其制造方法。(The purpose of the present invention is to provide a cell preparation containing MSCs that have a good therapeutic effect. A method of making activated MSCs is provided, including the use of a kitCulturing MSCs in a medium containing an activator derived from a mammalian fetal appendage with a cell culture carrier having a three-dimensional structure composed of fibers. In addition, a compound selected from the group consisting of p16 ink4a 、p14 ARF MSC therapeutic effect marker of the group consisting of CDK4, CDK6, RB and CD47, a method for determining therapeutic effect using the same marker, a method for determining the suitability of MSC for treatment for improving therapeutic effect, a cell preparation containing MSC and a method for producing the same.)

1. A method for producing an activated mesenchymal stem cell, comprising an activation step of treating a mesenchymal stem cell with an activator containing an extract from a mammalian fetal accessory as an active ingredient, wherein the activation step is a step of culturing the mesenchymal stem cell in the activator-containing medium using a cell culture carrier having a three-dimensional structure composed of fibers.

2. The method according to claim 1, wherein the cell culture carrier has an opening formed of fibers having an average fiber diameter of a nanometer to micrometer unit on a surface thereof which comes into contact with the cell.

3. The method according to claim 2, wherein the average diameter of the openings is 500nm to 1000. mu.m.

4. The production method according to any one of claims 1 to 3, wherein the cell culture carrier is a cell culture carrier containing nanofibers made of a biodegradable polymer.

5. The production method according to any one of claims 1 to 4, wherein the concentration of the activating agent in the medium is 1/10 to 1/100000 of the concentration of the activating agent in the medium in the step of activating the mesenchymal stem cells by culturing the mesenchymal stem cells using a cell culture carrier that does not have a three-dimensional structure composed of fibers.

6. The production method according to any one of claims 1 to 5, wherein the concentration of the activator in the medium is 0.01 to 500. mu.g/mL in terms of protein.

7. The production method according to any one of claims 1 to 6, wherein the number of subcultures of the cells in the activation step is 2 or less.

8. The production method according to any one of claims 1 to 7, wherein the mesenchymal stem cell is a bone marrow-derived mesenchymal stem cell.

9. The production method according to any one of claims 1 to 8, wherein the mesenchymal stem cell is a mesenchymal stem cell isolated from a subject having a disease.

10. A cell preparation containing mesenchymal stem cells, wherein the proportion of CD47 positive cells is 2% or less.

11. The cell preparation of claim 10, mesenchymal stem cells being activated mesenchymal stem cells produced by the method of any one of claims 1 to 9.

12. A method for producing a cell preparation containing mesenchymal stem cells, comprising a step of concentrating CD 47-negative mesenchymal stem cells from a mesenchymal stem cell cluster.

13. A marker for evaluating the therapeutic effect of mesenchymal stem cells, which is positively correlated with the therapeutic effect of mesenchymal stem cells, is selected from the group consisting of p16^ink4aAnd p14^ARFGroup (d) of (a).

14. A marker for evaluating the therapeutic effect of mesenchymal stem cells negatively correlated with the therapeutic effect of mesenchymal stem cells, selected from the group consisting of CDK4, CDK6, RB and CD 47.

15. A method of determining the therapeutic effect of mesenchymal stem cells, comprising: determining the presence of a compound selected from the group consisting of p16in the mesenchymal stem cells to be assayed^ink4a、p14^ARFA step of measuring the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD 47; a comparison step of comparing the measured expression level with an expression level in a control mesenchymal stem cell; and is selected from the group consisting of p16^ink4aAnd p14^ARFAnd a determination step of determining that the therapeutic effect of the mesenchymal stem cell to be tested is better than that of the control mesenchymal stem cell when the expression level of at least 1 gene or protein of the group consisting of the mesenchymal stem cell to be tested is higher than that of the control mesenchymal stem cell and/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is lower than that of the control mesenchymal stem cell.

16. The method of claim 15, wherein the first and second light sources are selected from the group consisting of,

the measuring step further comprises measuring the expression level of (A) and/or (B) in the mesenchymal stem cell to be measured,

(A) is at least 1 gene or protein selected from the group consisting of DNMT1, Nanog, SOX2, OCT4, IDO, TSG6, IL-6 and TERT,

(B) is at least 1 gene or protein selected from the group consisting of p53 and alpha-SMA,

the judging step is the following steps: in a group selected from p16^ink4aAnd p14^ARFWhen the expression amount of at least 1 gene or protein of the group consisting is more in the mesenchymal stem cell to be tested than in the control mesenchymal stem cell and/or when the expression amount of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is less in the mesenchymal stem cell to be tested than in the control mesenchymal stem cell, and when the expression amount of the gene or protein of (a) is more in the mesenchymal stem cell to be tested than in the control mesenchymal stem cell and/or when the expression amount of the gene or protein of (B) is less in the mesenchymal stem cell to be tested than in the control mesenchymal stem cell, it is judged that the therapeutic effect of the mesenchymal stem cell to be tested is better than that of the control mesenchymal stem cell.

17. A method for determining the therapeutic effect of mesenchymal stem cells, which comprises the step of measuring the ratio of CD47 positive cells in the mesenchymal stem cells in place of the expression level of CD47 gene or protein in the measuring step of the method according to claim 15 or 16; in the determination step, instead of the case where the expression level of the CD47 gene or protein is less in the mesenchymal stem cell to be tested than in the control mesenchymal stem cell, the therapeutic effect of the mesenchymal stem cell to be tested is determined to be better than that of the control mesenchymal stem cell when the proportion of the CD47 positive cells in the mesenchymal stem cell is lower in the mesenchymal stem cell to be tested than in the control mesenchymal stem cell.

18. The method of any one of claims 15 to 17, wherein the mesenchymal stem cells to be tested are mesenchymal stem cells cultured in a medium comprising an activator comprising an extract from a fetal accessory of a mammal as an active ingredient.

19. The method of any one of claims 15 to 18, wherein the mesenchymal stem cells to be tested are mesenchymal stem cells cultured using a cell culture carrier having a three-dimensional structure composed of fibers.

20. The method of any one of claims 15 to 19, wherein the mesenchymal stem cells to be tested are mesenchymal stem cells isolated from a subject to be subjected to a mesenchymal stem cell self-transplantation therapy.

21. The method of any one of claims 15 to 20, wherein the mesenchymal stem cells to be tested are bone marrow-derived mesenchymal stem cells.

22. A method of determining the suitability of a mesenchymal stem cell for a treatment for improving the therapeutic effect of a mesenchymal stem cell, comprising:

determining in the treated mesenchymal stem cells a selection from the group consisting of p16^ink4a、p14^ARFA step of measuring the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD 47;

a comparison step of comparing the measured expression level with an expression level in an untreated mesenchymal stem cell; and

in a group selected from p16^ink4aAnd p14^ARFAnd a determination step of determining that the mesenchymal stem cell is suitable for the treatment when the expression level of at least 1 gene or protein of the group consisting of the above-mentioned genes is higher in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell and/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is lower in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell.

23. The method of claim 22, wherein the first and second portions are selected from the group consisting of,

the measuring step further comprises measuring the expression amount of (A) and/or (B) in each of the treated mesenchymal stem cell and the untreated mesenchymal stem cell, wherein,

(A) is at least 1 gene or protein selected from the group consisting of DNMT1, Nanog, SOX2, OCT4, IDO, TSG6, IL-6 and TERT,

(B) is at least 1 gene or protein selected from the group consisting of p53 and α -SMA;

the judging step is the following steps: in a group selected from p16^ink4aAnd p14^ARFThe mesenchymal stem cell is judged to be suitable for the treatment if the expression amount of at least 1 gene or protein of the group consisting of (a) is more in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell and/or the expression amount of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is less in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell, and if the expression amount of the gene or protein of (a) is more in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell and/or the expression amount of the gene or protein of (B) is less in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell.

24. A method for determining the suitability of mesenchymal stem cells for treatment, which comprises the step of measuring the proportion of CD 47-positive cells in the mesenchymal stem cells in place of the expression level of the CD47 gene or protein in the measuring step of the method according to claim 22 or 23; in the determination step, instead of the case where the expression amount of the CD47 gene or protein is smaller in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell, if the proportion of CD 47-positive cells in the mesenchymal stem cell is lower in the treated mesenchymal stem cell than in the untreated mesenchymal stem cell, it is determined that the mesenchymal stem cell is suitable for the treatment.

25. The method according to any one of claims 22 to 24, which is a treatment of culturing mesenchymal stem cells in a medium containing an activator containing an extract from a fetal accessory of a mammal as an active ingredient.

26. The method according to any one of claims 22 to 25, wherein the treatment is a treatment of culturing mesenchymal stem cells using a cell culture carrier having a three-dimensional structure composed of fibers.

27. The method of any one of claims 22 to 26, wherein the mesenchymal stem cells are mesenchymal stem cells isolated from a subject to be subjected to a mesenchymal stem cell self-transplant therapy.

28. The method of any one of claims 22 to 27, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.

Technical Field

The present invention relates to a method for producing mesenchymal stem cells, a marker for evaluating the therapeutic effect of mesenchymal stem cells, a method for determining the suitability of mesenchymal stem cells for treatment for improving the therapeutic effect, and a cell preparation containing mesenchymal stem cells.

Background

Mesenchymal stem cells (hereinafter also referred to as MSCs) are multipotent and self-replicating stem cells, and have the ability to differentiate into Mesenchymal cells such as osteoblasts, chondrocytes, adipocytes and muscle cells, as well as into neural cells, hepatocytes and the like beyond the germ layer. In addition, MSCs are known to have paracrine effects and cell adhesion interactions brought about by self-produced humoral factors. Based on these effects, MSCs are thought to exhibit therapeutic effects on various diseases as a result of exhibiting an immunomodulatory ability such as a tissue and cell repair/regeneration ability and anti-inflammation ability.

Since MSCs are easily isolated and cultured and have high proliferation potency, the number of cells that can be transplanted can be ensured in a short time; (ii) capable of self-transplantation without immune rejection; the ethical problem is less; allografting is realistic because of low immunogenicity without pretreatment, etc.; therefore, as an ideal material for cell transplantation therapy, research is being conducted on the application of the material to the treatment of various diseases.

The present inventors found that: MSCs of a patient such as a diabetic patient are abnormal MSCs, and specifically, MSCs lose disease treatment effect or treatment effect is reduced from normal MSCs as a result of losing as many as the aforementioned abilities or these abilities are reduced from normal MSCs; extracts from mammalian fetal appendages are capable of restoring therapeutic efficacy by activating the aforementioned aberrant MSCs; and can realize autologous transplantation therapy using the activated MSCs, an activator for abnormal MSCs containing an extract from a mammalian fetal accessory as an active ingredient has been invented and applied for a patent (patent document 1). This activator is of great significance, especially in terms of being able to achieve MSC autologous transplantation also for patients in the necessary treatment phase.

The activator for abnormal MSCs described in patent document 1 can be produced as an extract from a mammalian fetal appendage, for example, human umbilical cord tissue or placental tissue. Fortunately, fetal appendages of mammals including humans are biological samples that are relatively easily obtained and can be expected to be supplied in a certain amount. However, the supply of mammalian fetal appendages and active agents extracted therefrom is hardly said to be sufficient for enabling MSC self-transplantation to patients for whom MSC cell transplantation therapy is expected to be applied, for example, a large number of diabetic patients who are continuously increasing worldwide.

Disclosure of Invention

Problems to be solved by the invention

According to the study of the present inventors, it was clarified that: the activator can not only improve the treatment effect of abnormal MSC, but also improve the treatment effect of MSC of healthy people; on the other hand, there are MSCs and the like, for which improvement in therapeutic effect is hardly observed even when treated with an activator. As described above, an activator containing an extract from a mammalian fetal appendage as an active ingredient is a valuable substance that needs to be effectively utilized, and even if it is a result, it is not preferable for MSC in which little improvement in therapeutic effect is observed. Further, even when MSC that does not show an improvement in therapeutic effect even by treatment with an activator is administered to a patient, the effect cannot be expected, and therefore, it is desired to confirm the activity before administration.

Accordingly, an object of the present invention is to provide a method for activating MSCs more efficiently in the production of MSCs activated by an activator containing an extract derived from a mammalian fetal appendage as an active ingredient. It is also an object of the present invention to provide a method for evaluating the therapeutic effect of MSCs, a method for determining whether MSCs isolated from a subject have suitability for treatment intended to improve the therapeutic effect, such as treatment with an activating agent, and a cell preparation containing MSCs having a good therapeutic effect.

Means for solving the problems

The present inventors have found that activated MSCs having a better therapeutic effect can be produced with a smaller amount of an activating agent by activating abnormal MSCs on a cell culture support having a three-dimensional structure composed of fibers, and that normal MSCs are also activated by culturing in the presence of an activating agent on the cell culture support. Further, the present inventors have analyzed the gene expression profile of MSCs with good therapeutic effects and have found specific gene expression changes. The following inventions have been made based on these findings.

(1) A method for producing activated MSCs, comprising an activation step of treating MSCs with an activator containing an extract derived from a mammalian fetal appendage as an active ingredient, wherein the activation step is a step of culturing MSCs in a medium containing the activator using a cell culture carrier having a three-dimensional structure composed of fibers.

(2) The production method according to (1), wherein the cell culture carrier has an opening formed of a fiber having an average fiber diameter of a nanometer to micrometer unit on a surface that comes into contact with the cell.

(3) The production method according to (2), wherein the average diameter of the openings is 500nm to 1000. mu.m.

(4) The production method according to any one of (1) to (3), wherein the cell culture carrier contains nanofibers made of a biodegradable polymer.

(5) The production method according to any one of (1) to (4), wherein the concentration of the activating agent in the medium is 1/10 to 1/100000 of the concentration of the activating agent in the medium in the step of culturing MSCs using a cell culture carrier having no three-dimensional structure composed of fibers.

(6) The production method according to any one of (1) to (5), wherein the concentration of the activator in the medium is 0.01 to 500. mu.g/mL in terms of protein.

(7) The production method according to any one of (1) to (6), wherein the number of subcultures of the cells in the activation step is 2 or less.

(8) The production method according to any one of (1) to (7), wherein the MSCs are bone marrow-derived MSCs.

(9) The production method according to any one of (1) to (8), wherein the MSC is an MSC isolated from a subject having a disease.

(10) A cell preparation containing MSC has a CD47 positive cell content of 2% or less.

(11) The cell preparation according to (10), wherein the MSC is an activated MSC produced by the method of any one of (1) to (9).

(12) A method for producing a cell preparation containing MSCs, comprising a step of concentrating CD 47-negative MSCs from an MSC cluster.

(13) A marker for evaluating MSC therapeutic effect positively correlated with MSC therapeutic effect selected from the group consisting of p16^ink4aAnd p14^ARFGroup (d) of (a).

(14) A marker negatively correlated with MSC therapeutic effect for evaluating MSC therapeutic effect, selected from the group consisting of CDK4, CDK6, RB and CD 47.

(15) A method of determining the efficacy of a treatment with MSCs, comprising: determination of MSC to be tested selected from the group consisting of p16^ink4a、p14^ARFA step of measuring the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD 47; a comparison step of comparing the expression level thus measured with the expression level in the control MSC; and is selected from the group consisting of p16^ink4aAnd p14^ARFAnd a determination step of determining that the therapeutic effect of the test MSC is better than that of the control MSC when the expression level of at least 1 gene or protein in the test MSC is higher than that in the control MSC and/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB, and CD47 is lower than that in the test MSC than that in the control MSC.

(16) The method according to (15), wherein,

the measuring step further comprises measuring the expression level of (A) and/or (B) in the MSC to be measured,

(A) is at least 1 gene or protein selected from the group consisting of DNMT1, Nanog, SOX2, OCT4, IDO, TSG6, IL-6 and TERT,

(B) is at least 1 gene or protein selected from the group consisting of p53 and α -SMA;

the judging step is the following steps: in a group selected from p16^ink4aAnd p14^ARFThe expression level of at least 1 gene or protein of the group consisting of more in the test MSCs than in the control MSCs and/or the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47And (B) determining that the therapeutic effect of the test MSC is better than that of the control MSC when the amount of the gene or protein expressed in the test MSC is less than that in the control MSC, when the amount of the gene or protein expressed in the test MSC is more than that in the control MSC, and/or when the amount of the gene or protein expressed in the test MSC is less than that in the control MSC.

(17) A method for determining the therapeutic effect of MSCs, which comprises measuring the proportion of CD47 positive cells in MSCs, instead of the expression level of CD47 gene or protein in the measurement step of the method of (15) or (16); in place of the case where the expression level of the CD47 gene or protein in the test MSC is less than that in the control MSC in the determination step, the treatment effect of the test MSC is determined to be better than that of the control MSC in the case where the proportion of CD47 positive cells in the MSC is lower than that in the control MSC.

(18) The method according to any one of (15) to (17), wherein the MSCs to be tested are MSCs cultured in a medium containing an activator containing an extract from a fetal accessory of a mammal as an active ingredient.

(19) The method according to any one of (15) to (18), wherein the MSCs to be tested are MSCs cultured using a cell culture carrier having a three-dimensional structure composed of fibers.

(20) The method according to any one of (15) to (19), wherein the MSC to be tested is an MSC isolated from a subject to be subjected to MSC autograft therapy.

(21) The method according to any one of (15) to (20), wherein the MSCs to be tested are bone marrow-derived MSCs.

(22) A method of determining the suitability of an MSC for a treatment for improving the therapeutic effect of an MSC, comprising: determining the concentration of selected from p16in the treated MSCs^ink4a、p14^ARFA step of measuring the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD 47; a comparison step of comparing the measured expression level with an expression level in an untreated MSC; and is selected from the group consisting of p16^ink4aAnd p14^ARFThe expression level of at least 1 gene or protein of the group consisting of more instances in treated MSCs than in untreated MSCs and/or the expression of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47And a determination step of determining that the MSC is suitable for the treatment when the amount of the MSC is smaller in the treated MSC than in the untreated MSC.

(23) The method according to (22), wherein,

the measuring step further comprises measuring the expression level of (A) and/or (B) in each of the treated MSC and the untreated MSC,

(A) is at least 1 gene or protein selected from the group consisting of DNMT1, Nanog, SOX2, OCT4, IDO, TSG6, IL-6 and TERT,

(B) is at least 1 gene or protein selected from the group consisting of p53 and α -SMA;

the judging step is the following step: in a group selected from p16^ink4aAnd p14^ARFWhen the expression level of at least 1 gene or protein of the group consisting of the gene or protein is more in the treated MSC than in the untreated MSC and/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is less in the treated MSC than in the untreated MSC, and when the expression level of the gene or protein of the aforementioned (a) is more in the treated MSC than in the untreated MSC and/or when the expression level of the gene or protein of the aforementioned (B) is less in the treated MSC than in the untreated MSC, the MSC is judged to be suitable for the treatment.

(24) A method for determining the suitability of MSC for treatment, which comprises the step of measuring the proportion of CD47 positive cells in MSC instead of the expression level of CD47 gene or protein in the measuring step of the method of (22) or (23); in the determination step, instead of the case where the expression level of the CD47 gene or protein is less in the treated MSCs than in the untreated MSCs, if the proportion of CD 47-positive cells in the MSCs is lower in the treated MSCs than in the untreated MSCs, the MSCs are determined to be suitable for the treatment.

(25) The method according to any one of (22) to (24), wherein the treatment is a treatment of culturing MSCs in a medium containing an activator containing an extract from a fetal accessory of a mammal as an active ingredient.

(26) The method according to any one of (22) to (25), wherein the treatment is a treatment of culturing MSCs using a cell culture carrier having a three-dimensional structure composed of fibers.

(27) The method according to any one of (22) to (26), wherein the MSC is isolated from a subject to be subjected to MSC autograft therapy.

(28) The method of any one of (22) to (27), wherein the MSCs are bone marrow-derived MSCs.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the method for producing MSCs of the present invention, MSCs can be activated with a much smaller amount of activating agent than in the past, and the consumption of precious activating agent can be reduced. The cell preparation of the present invention produced by the production method has a good therapeutic effect, and the number of cells necessary for therapy or the like can be reduced. The reduction in the number of cells to be administered brings about a reduction in the number of cell culture subcultures, resulting in the effects of shortening the time required for the production of a cell preparation and reducing the cost. Further, according to the therapeutic effect marker and the judgment method of the present invention, the therapeutic effect of MSC can be judged before administration to a subject. Furthermore, according to the method for determining the suitability of MSCs for treatment such as activation treatment, before the required amount of MSCs for treatment is prepared, the suitability of MSCs can be determined.

Drawings

Fig. 1 is a view showing the results of cluster analysis of gene expression profiles of MSC (hereinafter referred to as OA-MSC) of hip arthropathy patients cultured in the presence of an activating agent on a cell culture carrier having a three-dimensional structure composed of fibers (hereinafter also referred to as a three-dimensional culture carrier).

FIG. 2 is a graph showing the relative expression amounts of marker genes in OA-MSC cultured in the presence of an activating agent on a two-dimensional structure cell culture carrier of the prior art (hereinafter referred to as a two-dimensional culture carrier) and OA-MSC cultured in the presence of an activating agent on a three-dimensional culture carrier compared with the genes of OA-MSC cultured in the absence of an activating agent on a two-dimensional culture carrier.

FIG. 3 is an electron microscope observation photograph showing the appearance of OA-MSC cultured on a three-dimensional culture carrier and OA-MSC cultured on a two-dimensional culture carrier. The upper left corresponds to OA-MSC cultured on the three-dimensional culture carrier without addition of an activator (magnification 600 times), the upper right corresponds to OA-MSC cultured on the three-dimensional culture carrier with addition of an activator (magnification 200 times), the lower left corresponds to OA-MSC cultured on the two-dimensional culture carrier without addition of an activator (magnification 700 times), and the lower right corresponds to OA-MSC cultured on the two-dimensional culture carrier with addition of an activator (magnification 500 times).

FIG. 4 is a photograph of a high magnification electron microscope observation showing the appearance of OA-MSC cultured on a three-dimensional culture carrier and OA-MSC cultured on a two-dimensional culture carrier. The upper left corresponds to OA-MSC cultured on the three-dimensional culture carrier without addition of an activating agent (magnification 3500 times), the upper right corresponds to OA-MSC cultured on the three-dimensional culture carrier with addition of an activating agent (magnification 2500 times), the lower left corresponds to OA-MSC cultured on the two-dimensional culture carrier without addition of an activating agent (magnification 7000 times), and the lower right corresponds to OA-MSC cultured on the two-dimensional culture carrier with addition of an activating agent (magnification 8000 times).

FIG. 5 is a view showing the results of cluster analysis of the gene expression profiles of OA-MSC cultured on the cell culture carrier of the comparative example or other three-dimensional culture carriers in the presence of an activating agent, based on the profile of the three-dimensional culture carrier.

FIG. 6 is an electron microscope photograph (3500-fold magnification) showing the appearance of OA-MSC cultured by adding an activator to another three-dimensional culture carrier.

Fig. 7 is a graph showing the relative expression amounts of OCT4, SOX2, and TSG-6 genes in healthy human MSCs cultured in the presence of an activator on a two-dimensional culture carrier and healthy human MSCs cultured in the presence of an activator on a three-dimensional culture carrier compared to those genes in healthy human MSCs cultured in the absence of an activator on a two-dimensional culture carrier.

Fig. 8 is a graph showing the residual defect area on days 3 and 7 after skin excision of rats using a skin defect model administered with OA-MSC treated with an activating agent.

Fig. 9 is a photograph showing a skin defect part 7 days after skin excision of rats using a skin defect model of OA-MSC treated with an activating agent.

Fig. 10 is a graph showing the results of measurement of skin tissue rupture stress after 21 days of skin excision of rats using a skin defect model administered with OA-MSC treated with an activating agent.

Figure 11 is a graph showing the results of a learning test in the morris water maze using an alzheimer's disease model mouse dosed with OA-MSCs treated with an activator. The escape latency on the vertical axis represents the time for the mouse to reach the platform placed in the pool, and the horizontal axis represents the number of days from the start of the learning test.

Figure 12 is a graph showing the results of memory testing in the morris water maze using OA-MSC dosed with activator. The quadrant time on the vertical axis represents the proportion of time spent swimming in the 4 divided zones with the platform during the learning process relative to the total swimming time.

FIG. 13 is a graph showing the evolution of serum creatinine in diabetic nephropathy rats treated with an anticancer agent implanted with OA-MSC treated with an activating agent.

Fig. 14 is a graph showing the evolution of survival rate from diabetic nephropathy rats treated with an anticancer agent to 11 weeks after transplantation, in which OA-MSC treated with an activating agent was transplanted. The thick line represents the mean value of each group and the thin line represents the 95% CI (confidence interval) of each group.

Fig. 15 is a graph showing the results of cluster analysis on the gene expression profile of OA-MSC (n ═ 10).

Fig. 16 is a graph showing the results of cluster analysis on the gene expression profile of OA-MSC (n ═ 10).

FIG. 17 is a graph showing the calculated relative expression levels of each of OCT4, SOX2, NANOG, IDO and TSG6 and p16 for the gene expression levels in OA-MSC treated with an activator compared to the gene expression levels in untreated MSC^ink4aA graph showing the correlation between the relative expression levels of (a).

FIG. 18 is a graph showing the calculated p14 when the gene expression level in OA-MSC treated with activator was compared with that of untreated MSC^ARFRelative expression amount of p16^ink4aA graph showing the correlation between the relative expression levels of (a).

FIG. 19 shows OCT4, p1 in OA-MSC (BM-021, BM-022, BM-023)6^ink4aAnd a graph of the relative expression level of RB gene. In the figure, the OA-MSC treated with the activating agent is denoted as PE + or PE + MSC, and the untreated OA-MSC is denoted as PE-or PE-MSC (the same applies to the following figures).

FIG. 20 is a diagram showing OCT4, SOX2 and p16in OA-MSC (BM-010) treated with an activating agent on a three-dimensional culture support^ink4aGraph of relative expression of genes.

Fig. 21 is a graph showing the relative expression amount of CD47 gene in each of rheumatoid arthritis patient MSC (ra), old-aged MSC (aged), cirrhosis patient MSC, and young-aged MSC (young) treated with an activating agent on a three-dimensional culture carrier compared with that of MSC cultured in the absence of an activating agent on a two-dimensional culture carrier.

Fig. 22 is a histogram showing the amount of CD47 expression on the cell surface in untreated young and old MSCs, respectively.

Fig. 23 is a histogram showing the amount of CD47 expression on the cell surface in each of young and old MSCs treated with an activating agent on a three-dimensional culture carrier.

FIG. 24 shows p16in OA-MSC (BM-008, BM-021, BM-005)^ink4aGraph of relative expression of genes.

FIG. 25 is a graph showing the phagocytic ability of RAW264.7 cells co-cultured with OA-MSC (BM-008, BM-021, BM-005).

FIG. 26 is a graph showing the relative expression level of Fpr2(Formyl Peptide Receptor-2, Formyl Peptide Receptor) gene in RAW264.7 cells co-cultured with OA-MSC (BM-008, BM-021, BM-005).

FIG. 27 shows OCT4, SOX2 and p16in OA-MSC (BM-021)^ink4aGraph of relative expression of genes.

FIG. 28 is a graph showing the ratio of total macrophages (M Φ), M1 macrophages, and M2 macrophages relative to the total number of monocytes except for dead cells in multiple myositis model mice topically administered with OA-MSC (BM-021) or PBS treated or untreated with an activating agent.

FIG. 29 is a graph showing hind limb muscle function in multiple myositis model mice using topical administration of OA-MSC (BM-021) treated or untreated with an activator.

FIG. 30 is a diagram showing OCT4, SOX2 and p16in OA-MSC (BM-008)^ink4aGraph of relative expression of genes.

FIG. 31 is a graph showing muscle function in multiple myositis model mice systemically administered with OA-MSC (BM-008) or PBS treated or untreated with an activating agent.

FIG. 32 is a image of HE staining of muscle tissue of polymyositis model mice administered systemically with OA-MSC (BM-008) or PBS treated or untreated with an activating agent.

FIG. 33 is a graph showing the muscle cross-sectional area of multiple myositis model mice systemically administered OA-MSC (BM-008) or PBS treated with an activating agent or untreated.

FIG. 34 is a diagram showing OCT4, SOX2 and p16in OA-MSC (BM-021) treated with an activating agent on a two-dimensional culture carrier or a three-dimensional culture carrier^ink4aGraph of relative expression of genes.

FIG. 35 shows OCT4, SOX2, NANOG, p16in OA-MSC treated with activator after transplantation into diabetic nephropathy rats treated with anticancer agent^ink4a、p14^ARFGraph of the relative expression levels of the individual genes IDO, α -SMA, and TERT compared to those of MSCs cultured in the absence of the activator on a two-dimensional culture support.

FIG. 36 is a p16 showing CDKN2A knock-out OA-MSCs treated with activators on a three-dimensional culture support^ink4aAn image of the expression of (1).

Fig. 37 is a graph showing a staining image of muscle tissue laminin (left) and a muscle cross-sectional area (right) of a polymyositis model mouse locally administered in the triceps surae using CDKN2A knockout OA-MSC treated with an activator on a three-dimensional culture carrier.

Fig. 38 is a graph (left) showing the amount of change in wound area from day 3 to day 9 after skin excision in a skin defect model mouse administered with OA-MSC overexpressing CD47 and a photograph (right) of a representative example of a skin defect portion at day 9 after skin excision.

FIG. 39 is a graph showing the results of a learning test in the Morris water maze in Alzheimer's disease model mice dosed with 2 OA-MSCs with different CD47 positive cell rates.

FIG. 40 is a graph showing the results of a memory test in the Morris water maze in Alzheimer's disease model mice dosed with 2 OA-MSCs with different CD47 positive cell rates.

FIG. 41 is a graph showing the relative expression amounts of OCT4, SOX2, and TSG-6 genes in OA-MSCs cultured on a two-dimensional culture carrier or a three-dimensional culture carrier in the presence of other activators and OA-MSCs cultured on a three-dimensional culture carrier in the absence of activators in comparison with those genes in healthy human MSCs cultured on a two-dimensional culture carrier in the absence of activators, which differ in the extraction method.

FIG. 42 is a diagram showing that p16in OA-MSC cultured on a two-dimensional culture carrier or a three-dimensional culture carrier in the presence of other activating agents and OA-MSC cultured on a three-dimensional culture carrier in the absence of an activating agent differs in the extraction method^ink4aGraph of the relative expression amount of a gene compared to that of a healthy human MSC cultured in the absence of an activating agent on a two-dimensional culture carrier.

Detailed Description

Method for producing activated MSC

A first aspect of the present invention relates to a method for producing activated MSCs, the method comprising an activation step of treating MSCs with an activator containing an extract derived from a mammalian fetal appendage as an active ingredient, wherein the activation step is a step of culturing MSCs in a medium containing the activator using a cell culture carrier having a three-dimensional structure composed of fibers.

The MSC used in the present invention may be an MSC isolated from a healthy subject or an MSC isolated from a diseased subject. The MSC used in the present invention may be a cell induced by differentiation of a pluripotent stem cell such as an artificial pluripotent stem cell (iPS cell), an embryonic stem cell (ES cell), an embryonic tumor cell (EC cell), or an embryonic germ stem cell (EG cell).

The terms "subject", "MSC isolated from a subject with a disease" and "extract from a mammalian fetal accessory" in the present specification are all interpreted to have the same meaning as the terms described in the international publication WO2015/137419 pamphlet of patent document 1 and the U.S. patent application publication US2017/0071984 publication corresponding thereto. These documents are incorporated in their entirety into the present specification by reference, and the summary of the meanings of these documents and the terms in the present specification is shown below.

The "subject" means any animal having MSCs, preferably a mammalian subject, for example, a primate such as a human and chimpanzee, a rodent such as a mouse, rat, guinea pig, and hamster, an artiodactyla such as a cow, goat, sheep, and pig, a perissodactyla such as a horse, a rabbit, a dog, and a cat, and more preferably a human subject.

As described in patent document 1, it is known that MSC treatment effects of some disease subjects and aging subjects are inferior to those of healthy people, and even if MSC is transplanted directly to these subjects, a good treatment effect cannot be expected. On the other hand, according to the manufacturing method of the first aspect, even such MSCs with poor therapeutic effects can be activated, and the therapeutic effects can be improved. The disease of a subject having MSCs with poor therapeutic effect is a chronic disease, and an example thereof is described as a disease in which MSCs are abnormal in patent document 1.

In consideration of safety in the subsequent cell transplantation therapy, MSCs are preferably collected from an individual of the same species as or a closely related species as an individual to which cells are administered. For example, when a human individual is transplanted with cells, it is preferable to use cells collected from the same human, and it is more preferable to use autologous MSCs, which are cells collected from the same human individual to which the drug is administered.

The MSCs used in the present invention can be collected from a sample such as bone marrow fluid, adipose tissue, fetal accessory tissue, dental pulp, or the like of a subject by a general method. For example, when bone marrow fluid is used as a sample, MSCs can be isolated by a known method such as density gradient centrifugation or bone marrow seeding. In a preferred embodiment of the invention, the MSCs are bone marrow derived MSCs.

The "extract of a mammalian-derived fetal appendage" is an extract prepared by directly or by cutting or crushing a fetal appendage, preferably an umbilical cord tissue, a placental tissue, or an egg membrane, which is delivered from a mother as a post-product after delivery of a mammalian, preferably a human fetus or is taken out of the mother by caesarean section, and soaking the fetal appendage in an extraction medium such as distilled water, physiological saline, phosphate buffered physiological saline, or a medium generally used in cell culture. The extract is particularly preferably free of cells having proliferative capacity of mammalian origin as donor. Specific extraction operations and conditions may follow those described in patent document 1.

In addition, the "extract of fetal appendages derived from mammals" may be prepared by subjecting the fetal appendages to a treatment generally used by those skilled in the art, for example, hydrolysis using an acid or an enzyme, or the like, when extracting a physiologically active substance from the fetal appendages, typically from the placenta. Examples of the extract include "meisimen" which is a human placenta extract sold by meisimen pharmaceutical corporation, "LAENNEC" which is a human placenta extract sold by natural biological preparations of kakkimen corporation, other commercially available placenta preparations, and various commercially available products called placenta extracts.

The "activator containing an extract derived from a fetal appendage of a mammal as an active ingredient" in the present invention refers to a preparation for enhancing the therapeutic effect of MSC containing the aforementioned extract as an active ingredient, and hereinafter, it is referred to as "activator". Here, "activation" means that MSCs that received a certain treatment show better therapeutic effects than those without treatment.

In the production method of the first aspect, the activation step is performed by culturing MSCs in vitro in a medium containing the aforementioned activating agent using a cell culture carrier having a three-dimensional structure composed of fibers. Specifically, it is performed by culturing MSCs in the presence of an activating agent using a three-dimensional culture support as a scaffold.

In a preferred embodiment, the three-dimensional culture support is composed of fibers having an average fiber diameter of nanometer (nm) to micrometer (μm) units. Here, the "average fiber diameter" is an arithmetic average of fiber diameters measured as the length in a direction perpendicular to the fiber length direction when the culture carrier is observed from the cell-adherent surface side, typically from above. In a more preferred embodiment, the three-dimensional culture carrier has openings formed by fibers having an average fiber diameter of a unit of nanometer (nm) to micrometer (μm) on a surface that comes into contact with cells. Here, the "opening" refers to a depression formed by the above-mentioned fiber and existing on the contact surface between the carrier and the cell. Wherein, unless otherwise specified, the average in this specification means a number average.

When the fibers are in contact with each other, the average diameter of the openings is the average of the diameters of the patterns outlined by the fibers when the culture carrier is viewed from above. The diameter of the figure corresponds to the arithmetic average of the lengths of the diagonal lines from the vertices in the case where the figure is a polygon, to the diameter in the case where the figure is a circle, and to the major diameter in the case where the figure is an ellipse or a shape similar thereto.

When the fibers do not contact each other, the average diameter of the openings means an average flow pore diameter obtained by a method specified in ASTM-F316, and can be measured by an average flow point method using, for example, a pore diameter meter (manufactured by coulter corporation).

The average fiber diameter of the fibers constituting the three-dimensional culture carrier may be in the range of nm to μm unit, preferably 10nm to 500 μm, and more preferably 10nm to 300 μm. In a specific embodiment, the average fiber diameter may be, for example, in the range of 10nm to 1 μm, 100nm to 1 μm, 500nm to 1 μm, 1 μm to 10 μm, 1 μm to 100 μm, 1 μm to 300 μm, or 1 μm to 500 μm, preferably in any one of the ranges of 10nm to 1 μm, 1 μm to 10 μm, or 10 μm to 300 μm. In the present invention, fibers generally called nanofibers can also be used.

In the three-dimensional culture carrier having openings on the surface to which cells adhere, the average diameter of the openings may be 500nm to 1000. mu.m, preferably 700nm to 600. mu.m, more preferably 900nm to 400. mu.m. In a specific embodiment, the average diameter of the openings may be, for example, in the range of 500nm to 100. mu.m, 5 μm to 100. mu.m, 10 μm to 100. mu.m, 20 μm to 100. mu.m, 100 μm to 200. mu.m, 100 μm to 400. mu.m, or 100 μm to 600. mu.m, and preferably in the range of 500nm to 100. mu.m, or 100 μm to 400. mu.m.

In a particular embodiment, the three-dimensional culture carrier has a void fraction of 60% or more, preferably 70% or more, more preferably 75% or more, and particularly preferably 80% or more.

In another embodiment, the average area of the openings of the three-dimensional culture carrier is 0.1 to 100 μm²Preferably 0.2 to 60 μm²More preferably 0.5 to 30 μm². When the opening is a hole, the opening area corresponds to the hole area.

The three-dimensional culture carrier has a structure in which fibers are three-dimensionally stacked, that is, fibers are stacked in a three-dimensional direction, and the arrangement of the fibers may be regular or irregular, and the fibers may or may not be bonded to each other.

The three-dimensional culture carrier may have a three-dimensional structure composed of fibers on the cell adhesion surface, or may have a portion that does not adhere to cells, typically a member that serves as a base under the three-dimensional structure composed of fibers. The base member may be any structure as long as it can support the three-dimensional structure, and may be, for example, a nonwoven fabric, a knitted fabric, a woven fabric, a porous scaffold material, or the like.

The cell adhesion surface in the three-dimensional culture carrier may be a portion whose main portion has a three-dimensional structure composed of fibers, and specifically, the portion having a three-dimensional structure composed of fibers may occupy 50% or more of the area of the culture carrier when the culture carrier is viewed from the cell adhesion surface side, typically from above. Therefore, a part of the cell-adherent surface of the three-dimensional culture carrier may include a part other than the "three-dimensional structure composed of fibers", for example, a flat film-like part having no three-dimensional structure, or the like in an area of the part of the cell-adherent surface of the three-dimensional culture carrier of less than 50%, preferably less than 40%, and more preferably less than 30%.

The material for forming the fibers is not particularly limited, and examples thereof include polymer compounds such as polyvinyl fluoride and polystyrene, inorganic compounds such as silica, and biodegradable polymers. Examples of the biodegradable polymer include those having biocompatibility and capable of maintaining a three-dimensional structure on a culture carrier for a desired period of time, for example, the synthetic polymer materials include the copolymers described above such as polyglycolic acid, polylactic acid, polyethylene glycol, polycaprolactone, polydioxanone, and other lactic acid-glycolic acid copolymers, the inorganic materials include β -tricalcium phosphate and calcium carbonate, and the natural polymer materials include collagen, gelatin, alginic acid, hyaluronic acid, agarose, chitosan, fibrin, fibroin, chitin, cellulose, and silk.

The shape of the three-dimensional culture carrier is not limited as long as the cells can be brought into contact with the three-dimensional structure composed of fibers at the time of culture. The three-dimensional culture carrier may be in the form of an insert to be used by being installed in the cell culture container, or may be in the form of a three-dimensional structure made of fibers integrally molded on the inner surface of the cell culture container, for example, the bottom surface of the well.

Examples of the three-dimensional culture carrier usable in the present invention include VECELL (registered trademark) (polytetrafluoroethylene, average fiber diameter: < 1 μm, average pore area: 1 to 20 μm) available from Vessel corporation²Average diameter of opening portion: 20-100 μm, porosity: 80 to 90%), cellded (registered trademark) of solieria corporation (high purity silica fiber, average fiber diameter: 1 μm, mean flow pore size: 7-8 μm, porosity: > 95%), 3D Insert-PS series of 3D Biotek (polystyrene fibers, average fiber diameter: PS-200 and PS-400 are in the ranges of 150 and 300 μm, and the average diameter of the openings: 200 μm for PS-200 and 400 μm for PS-400), a cell culture substrate described in the pamphlet of international publication WO2014/196549, a cell culture substrate (biodegradable polymer, average fiber diameter 50nm to 5 μm) described in the pamphlet of international publication WO2016/068266 and the japanese patent application publication US2017/319747 corresponding thereto, and a cell culture substrate (polyglycolic acid, average fiber diameter: 345. + -. 91nm, mean pore sizeArea: 0.68 +/-0.02 mu m²Average diameter of opening portion calculated from average hole area: 0.93 μm ((0.68 μm)²/π)^1/2) X 2), neoviil and neoviil NANO (county is corporation), and the like. Each of the above documents is incorporated by reference in its entirety into this specification.

The culturing of MSCs on the three-dimensional culture carrier is performed for 24 to 144 hours, preferably 24 to 96 hours, and more preferably 48 to 96 hours. The culture temperature and gas concentration in the activation step may be in the range of the temperature and gas concentration generally used in MSC culture, for example, 25 to 37 ℃, preferably 30 to 37 ℃, more preferably 37 ℃, and the oxygen concentration is, for example, 2 to 30%, preferably 2 to 20%. The activation treatment may be performed a plurality of times before sufficient activation is achieved.

The final concentration of the activating agent in the medium in the activation step may be 0.01. mu.g/mL-500. mu.g/mL, preferably 0.02. mu.g/mL-300. mu.g/mL, 0.03. mu.g/mL-200. mu.g/mL, more preferably 0.04. mu.g/mL-100. mu.g/mL, in terms of protein, and in a specific embodiment may be 0.05. mu.g/mL-10. mu.g/mL. As is apparent from a comparison of the more preferable concentration of the activator exemplified in patent document 1, which is 0.1mg/mL (100. mu.g/mL) to 5mg/mL (5000. mu.g/mL), with that of the activator in the above-mentioned specific embodiment, the use of the three-dimensional culture carrier can reduce the concentration of the activator to 1/10 to 1/100000 in the past. The lower limit of the concentration range of the activating agent is, for example, 1/100000, 1/50000, 1/20000, 1/10000, 1/5000 or 1/2000 which is the concentration used when a conventional two-dimensional culture carrier is used, and the upper limit of the concentration range of the activating agent is, for example, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500 or 1/1000 which is the concentration used when a conventional two-dimensional culture carrier is used.

As the medium, a medium generally used for MSC culture, for example, α -MEM, DMEM, etc. can be used. These media may contain various components necessary for MSC growth, such as serum components, so long as activation is not hindered.

As shown in the following examples, MSCs cultured in the presence of an activating agent on a three-dimensional culture carrier have a better therapeutic effect than MSCs cultured in the presence of an activating agent on a two-dimensional culture carrier composed of polystyrene or the like, such as a culture carrier not having a three-dimensional structure composed of fibers, for example, Corning (registered trademark) Costar (registered trademark) cell culture plate (Thermo Fisher Science corporation). Since the effect can be expected even with a smaller number of cells if the therapeutic effect is better, the number of MSCs subculture in the activation step of the present invention can be smaller than that in the activation step of culturing MSCs using a cell culture carrier having no three-dimensional structure composed of fibers. The number of passages of MSCs in the activation step is preferably 2 or less, may be 1, and may be 0. The number of the secondary generations of the activated MSC produced by the production method of the first embodiment is preferably 3, that is, P3 or less, and may be P2, and further may be P1.

Whether or not the MSC is activated can be determined according to the method and criteria described in patent document 1. For example, in an evaluation system reflecting diseases such as disease model animals and cells derived from disease model animals, when the therapeutic effect of MSC cultured in the presence of an activating agent on a three-dimensional culture carrier (hereinafter also referred to as "3D activation-treated MSC") is better than that of MSC cultured in the absence of an activating agent on a two-dimensional culture carrier (hereinafter also referred to as "comparative MSC"), it is judged that the 3D activation-treated MSC is activated.

In addition, the activation can also be judged by using evaluation indexes such as the forms of cells and intracellular organelles, the cell proliferation ability, the differentiation ability, and the protein expression levels, the gene expression levels, and the extracellular secretion levels of various factors known as markers of MSCs having therapeutic effects, such as proliferation factors, differentiation-related factors, and cytokine-chemokines.

For example, when the cell proliferation ability or differentiation ability of the 3D-activated MSC is higher than that of the target MSC, the 3D-activated MSC may be judged to have a good therapeutic effect, i.e., to be activated.

Furthermore, when the expression level of a gene or protein of a stem cell-related factor (e.g., DNMT1, Nanog, SOX2, OCT4, etc.), an immunomodulatory-anti-inflammatory function-related factor (e.g., IDO, TSG6, IL-6, etc.), or a telomerase activity-related factor (e.g., TERT, etc.) is higher in 3D-activated MSCs than in comparison-target MSCs, or when the expression level of a gene or protein of a cell aging-related factor (e.g., P53, etc.) or a cytoskeleton-related factor (e.g., α -SMA, etc.) is lower in 3D-activated MSCs than in comparison-target MSCs, it can be determined that the 3D-activated MSCs are therapeutically effective, i.e., activated.

Further, when an increase in the cell thickness of MSCs, an increase in the number of processes, promotion of network structure formation, promotion of secretory vesicle formation, and the like are observed after 3D activation treatment, it can also be judged that the 3D activation-treated MSCs have a good therapeutic effect, i.e., are activated.

Furthermore, as described below, p16 having a high positive correlation with the therapeutic effect of MSC, which was newly found by the present inventors^ink4aAnd p14^ARFSimilarly, the inventors of the present invention newly found that gene or protein expression of CDK4, CDK6, RB and CD47, which have high negative correlation with the therapeutic effect of MSCs, can be used as an index to determine whether the therapeutic effect of 3D-activated MSCs is better than that of target MSCs, that is, whether 3D-activated MSCs are activated.

Activated MSCs may be maintained in an undifferentiated state, or may also differentiate into desired cells. The undifferentiated state of MSCs can be maintained by culturing MSCs in a medium suitable for maintaining the undifferentiated state, for example, in HyClone advanced Stem Cell Expansion Kit (semer femaly Technology), mesncult MSC basic medium (Stem Cell Technology), genomic cells media (dvbiologics), MSC-specific medium Kit (MSCGM bulkkit, dragon sand), or the like. In addition, differentiation of MSCs can be performed by a generally known method such as culture in a differentiation-inducing medium to which a factor having an action of inducing differentiation into desired cells is added. For example, in differentiation into osteoblasts, Bone Morphogenetic Proteins (BMPs) 4, BMP2, and the like are used as differentiation-inducing factors; for differentiation into adipocytes, dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and the like are used as differentiation-inducing factors.

In addition, MSCs can be stored by a general method such as cryopreservation before and after treatment such as activation, growth culture, and differentiation induction culture. For example, activated MSCs can be grown by culturing, then stored in separate containers with a constant number of cells, and thawed to a desired dose for administration.

MSC treatment effect marker and MSC treatment effect judgment method

Another mode of the present invention relates to a marker for evaluating the therapeutic effect of MSC, which is positively correlated with the therapeutic effect of MSC, and is selected from the group consisting of p16^ink4aAnd p14^ARFGroup (d) of (a). Furthermore, a further another mode of the present invention relates to a marker for evaluating MSC therapeutic effect negatively correlated with MSC therapeutic effect, selected from the group consisting of CDK4, CDK6, RB and CD 47.

p16^ink4aIs a cyclin-dependent kinase inhibitor having an ankyrin repeat. Known as p16^ink4aIs a cancer suppressor, and is also known as an aging-related gene whose expression level increases when cells are damaged by DNA due to the fact that they reach the division life, exposure to carcinogenic stress, etc. (E.Hara et al, mol.cell.biol., 1996, Vol.16, p.859-867; B.G.Childs et al, Nature Medicine, 2015, Vol.21, p.1424-1435).

p16^ink4aTogether with CDK4 and CDK6 as cyclin-dependent kinases, RB (retinoblastoma protein, the gene encoding RB is also called Rb) as cell cycle regulator, called p16^ink4a-the signalling pathway of the RB pathway. In this pathway, p16^ink4aBinds specifically to CDK4/6, blocking its action. Inhibition of CDK4/6 increased active dephosphorylated RB, which stopped the cell cycle. Hereinafter, these factors are also collectively referred to as p16^ink4a-an RB path factor.

Human p16^ink4aThe base sequences of genes and mRNAs translated into proteins are registered with the databases GenBank/NCBI under the accession numbers NM-000077 and NM-001195132, and the amino acid sequences of proteins are registered with the databases GenPept (NCBI Protein database) under the accession number NP-000068.

The base sequences of the genes of human CDK4 and CDK6 and mRNA translated into Protein are registered in the database GenBank/NCBI with accession numbers NM _000075 and NM _001259, and the amino acid sequence of Protein is registered in the database genpept (NCBI Protein database) with accession numbers NP _000066 and NP _001138778 (or NP _ 001250).

The base sequence of the gene of human RB and mRNA translated into protein is registered with the database GenBank/NCBI under the accession number NM-000321, and the amino acid sequence of protein is registered with the database GenPept (NCBIProtein database) under the accession number NP-000312.

p14^ARFIs p16^ink4aThe splice mutant of (1), both encoded by the CDKN2A gene. p14^ARFIs a p 53-stabilized protein having a function of inhibiting the degradation of p53 by binding to MDM2, and p16 is known^ink4aIt is also a cancer suppressor.

Human p14^ARFThe base sequences of the gene and mRNA translated into protein are registered in the database GenBank/NCBI under the accession number NM-058195, and the amino acid sequence of the protein is registered in the database GenPept (NCBIProtein database) under the accession number NP-478102.

CD47 (also known as integrin-associated protein (IAP)) is a transmembrane-type protein belonging to the immunoglobulin superfamily. It is known that CD47 expressed on the cell membrane of normal cells binds to a signal-regulatory protein alpha (SIRP alpha) on the cell membrane of macrophages, thereby inhibiting phagocytosis of macrophages. It is believed that to avoid phagocytosis by macrophages, it is preferred that the cells used in cell transplantation therapy express CD 47. In addition, CD47 was reported to enhance the homing and tissue repair effects of MSCs (Int JClin Exp Pathol.2015, 8 (9): 10555-.

The base sequences of the human CD47 gene and mRNA translated into Protein are registered in the database GenBank/NCBI under the accession numbers NM-001777 and NM-198793, and the amino acid sequence of the Protein is registered in the database GenPept (NCBI Protein database) under the accession number NP-001768.

Surprisingly, as a result of analyzing the gene expression profile of activated MSC, it was found that p16 was treated by activation^ink4a-a change in the expression of an RB pathway factor, in particular p16^ink4aTable of genesIncrease in expression and p16^ink4aReduction in gene expression levels of CDK4, CDK6, and RB as downstream factors, and the like. Furthermore, p14^ARFThe gene expression level of (2) and p16^ink4aAlso, an increase was shown, and on the other hand, the expression level of CD47 gene was shown to decrease. Furthermore, it was also confirmed experimentally that p16 as described above did not occur in MSCs in which no increase in therapeutic effect was observed even in the activation treatment^ink4a-RB pathway factor, p14^ARFAnd altered expression of CD 47.

Furthermore, the present inventors confirmed that the expression levels of genes involved in the therapeutic effects of MSC, such as OCT4, SOX2 and Nanog, which are factors involved in stem cell-mediated function, IDO and TSG-6, which are factors involved in immunoregulatory-anti-inflammatory function, were increased in activated MSC, and confirmed that p16^ink4aThe expression of the genes (a) has a very high positive correlation with the expression of these genes. p16^ink4aIs also related to p14^ARFIs positively correlated with gene expression.

In addition, it was confirmed that p16 was similar to the gene related to the therapeutic effect described above^ink4aAnd p14^ARFThe expression level was increased in MSCs with good therapeutic effects, and it was also confirmed that CD47 was reduced in MSCs with good therapeutic effects. Further, it was also confirmed by experiment that p16 was deleted^ink4aAnd p14^ARFThe therapeutic effect of MSCs of (a), forced expression of CD47 was reduced.

Based on these results, p16^ink4aAnd p14^ARFCan be used as a marker for evaluating the therapeutic effect of MSC which is positively correlated with the therapeutic effect of MSC on diseases, and p16^ink4aCan be used as a marker for evaluating the therapeutic effect of MSCs in negative correlation with the therapeutic effect of MSCs against disease. In addition, the therapeutic effect of MSCs can be assessed using these markers.

Yet another aspect of the present invention relates to a method of determining a therapeutic effect of MSCs, comprising: determination of MSC to be tested selected from the group consisting of p16^ink4a、p14^ARFA step of measuring the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD 47; for the measured expression amount and the pairA comparison step of comparing the expression levels of the MSCs; and is selected from the group consisting of p16^ink4aAnd p14^ARFAnd a determination step of determining that the therapeutic effect of the test MSC is better than that of the control MSC when the expression level of at least 1 gene or protein in the test MSC is higher than that of the control MSC and/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB, and CD47 is lower in the test MSC than that of the control MSC.

The method for judging the therapeutic effect comprises measuring the concentration of the MSC selected from the group consisting of p16^ink4a、p14^ARFAnd (ii) measuring the expression level of at least 1, preferably 2, 3 or all of the genes or proteins in the group consisting of CDK4, CDK6, RB and CD47 (hereinafter also referred to as therapeutic effect markers).

The MSC to be tested may be any MSC as long as it is desired to judge the therapeutic effect. An example of the MSC to be tested is an MSC destined for use in cell transplantation therapy. In the case of intended use in the self-transplant therapy, the MSC to be tested is an MSC isolated from the subject to be subjected to the self-transplant therapy, preferably of bone marrow origin. By judging the therapeutic effect of these MSCs before transplantation, unnecessary cell transplantation to a patient can be avoided, and the success rate of cell transplantation therapy can be improved. In a specific embodiment, the MSC to be tested may be an MSC subjected to a treatment for improving the therapeutic effect, and examples of the MSC include an MSC cultured in a medium containing an activating agent, i.e., an activated treatment, and an MSC cultured using a cell culture carrier having a three-dimensional structure composed of fibers.

The expression level of the gene or protein of the therapeutic effect marker in MSC can be measured by various methods known to those skilled in the art, which can measure the expression level of the gene or protein having a known base sequence or amino acid sequence.

The expression amount of the marker protein can be measured by a known method such as direct competition method, indirect competition method, ELISA such as sandwich method, RIA method, in situ hybridization, immunoblot analysis, western blot analysis, and tissue array analysis, using an antibody specific to the marker protein. In this case, the specific antibody is not limited by the species of the animal from which it is derived,the antibody may be either a polyclonal antibody or a monoclonal antibody, or may be a partial fragment including a full-length immunoglobulin antibody, a Fab fragment, or a F (ab') 2 fragment. Specific antibodies include fluorescent substances (e.g., FITC, rhodamine, phalloidin, etc.), colloidal particles of gold, etc., fluorescent microbeads of Luminex (registered trademark), etc., heavy metals (e.g., gold, platinum, etc.), pigment proteins (e.g., phycoerythrin, phycocyanin, etc.), radioisotopes (e.g., phycoerythrin, phycocyanin, etc.), and the like³H、¹⁴C、³²P、³⁵S、¹²⁵I、¹³¹I, etc.), enzymes (e.g., peroxidase, alkaline phosphatase, etc.), biotin, streptavidin, and other labeling compounds.

The measurement of the expression amount of a marker gene can be performed by a PCR method using a primer nucleic acid containing an appropriate base sequence designed based on the base sequence thereof, particularly a real-time PCR method, a digital PCR method capable of absolute quantification of nucleic acid, a hybridization method using a probe nucleic acid containing a base sequence capable of hybridizing with the base sequence of mRNA thereof under stringent conditions, a microarray method using a chip on which a probe nucleic acid containing a base sequence capable of hybridizing with the base sequence of mRNA thereof is immobilized, an RNA sequencing method, and other known methods capable of detecting the expression of the marker gene. The primer nucleic acid or the probe nucleic acid may be labeled with a fluorescent substance, a radioisotope, an enzyme, biotin, streptavidin, or another labeling compound, depending on the method used.

The method for judging a therapeutic effect according to the above aspect further includes a comparison step of comparing the measured expression level with the expression level in the control MSC. Here, the control MSC is an MSC as a criterion for judging the therapeutic effect. For example, in the case where the MSC to be tested is an MSC that is intended to be used in cell transplantation therapy, as the control MSC, an MSC having the lowest level of therapeutic effect that can be used in cell transplantation therapy can be used. The gene or protein expression level of the therapeutic effect marker in the control MSC may be measured simultaneously with the MSC to be tested, or may be prepared in advance.

The method for judging a therapeutic effect according to the above aspect further includes the following judging step: in a group selected from p16^ink4aAnd p14^ARFAnd/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is lower in the MSC to be tested than in the control MSC, determining that the therapeutic effect of the MSC to be tested is better than that of the control MSC.

In the determination step, the therapeutic effect marker measured in the measurement step is p16^ink4aOr p14^ARFIn the case of (2), p16^ink4aOr p14^ARFWhen the expression level of the gene or protein of (a) is higher in the MSC to be tested than in the control MSC, it can be judged that the treatment effect of the MSC to be tested is better than that of the control MSC. In addition, in the case where the therapeutic effect marker measured in the measurement step is CDK4, CDK6, RB or CD47, when the expression level of these genes or proteins is less in the MSC to be tested than in the control MSC, it can be judged that the therapeutic effect of the MSC to be tested is better than that of the control MSC.

In the case where a plurality of treatment effect markers are used to judge the treatment effect of MSCs, clustering analysis may also be used. Specifically, the relative expression data of each therapeutic effect marker measured in the MSC to be tested is used for cluster analysis together with the relative expression data of each therapeutic effect marker in the positive control MSC having a good therapeutic effect, that is, the MSC having a sufficient therapeutic effect for the level of the cell transplantation therapy, and the negative control MSC having a poor therapeutic effect, that is, the MSC having only a insufficient therapeutic effect for the level of the cell transplantation therapy, and the MSC to be tested and the positive control MSC can be judged to have a good therapeutic effect when they belong to the same cluster, and can be judged to have a poor therapeutic effect when they belong to the same cluster as the negative control MSC.

The clustering analysis can be performed by using statistical software such as R version 3.0.1 (variance R programming tools for clustering data; gplots, etc.) by using an appropriate clustering method, for example, a layering method such as a Ward method, a small group averaging method, etc.

Furthermore, the present inventors have also confirmed that, among MSCs having a good therapeutic effect, in addition to the above-described therapeutic effect markers, the expression levels of DNMT1, Nanog, SOX2, and OCT4 genes, TERT gene which is considered to be a gene related to telomerase activity, and the like, which are stem cell markers, are higher than those of untreated MSCs, and that the expression levels of p53, which is considered to be a gene related to cell aging, and α -SMA, which is considered to be a gene related to cytoskeleton, are lower than those of untreated MSCs. Therefore, in the measurement step, in addition to the gene or protein expression level of the therapeutic effect marker, the expression level of (A) and/or (B) is measured, wherein (A) is at least 1, preferably 2, 3, 4, 5, 6, 7 or all of the genes or proteins selected from the group consisting of DNMT1, Nanog, SOX2, OCT4, IDO, TSG6, IL-6 and TERT, and (B) is at least 1, preferably all of the genes or proteins selected from the group consisting of p53 and α -SMA, and the result thereof is combined with the change in the gene or protein expression level of the therapeutic effect marker in the judgment step, specifically,

in a group selected from p16^ink4aAnd p14^ARFThe present invention can also be said to be effective in the treatment of the MSC when the expression level of at least 1 gene or protein of the group consisting of the gene or protein is higher in the MSC to be tested than in the control MSC and/or the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is lower in the MSC to be tested than in the control MSC, and when the expression level of the gene or protein of the above-mentioned (a) is higher in the MSC to be tested than in the control MSC and/or the expression level of the gene or protein of the above-mentioned (B) is lower in the MSC to be tested than in the control MSC.

Further, the present invention also includes the following method: in the measurement step of the method for determining a therapeutic effect according to the above-described embodiment, the percentage of CD 47-positive cells in MSC is measured instead of the expression level of CD47 gene or protein; in the determination step, in place of the case where the expression level of the CD47 gene or protein is less in the test MSC than in the control MSC, the treatment effect of the test MSC is determined to be better than that of the control MSC when the proportion of CD47 positive cells in the MSC is lower in the test MSC than in the control MSC.

The proportion of CD 47-positive cells in MSCs can be determined by labeling MSCs with a labeled antibody against CD47, using isotype controls or the like as negative controls, and analyzing by flow cytometry or the like.

Adaptive determination of MSC for treatment to improve therapeutic EffectMethod of producing a composite material

A further aspect of the present invention relates to a method of determining the suitability of an MSC for a treatment for improving the therapeutic effect of the MSC, comprising: determining the concentration of selected from p16in the treated MSCs^ink4a、p14^ARFA step of measuring the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD 47; a comparison step of comparing the measured expression level with an expression level in an untreated MSC; and is selected from the group consisting of p16^ink4aAnd p14^ARFAnd a judging step of judging that the MSC is suitable for the treatment when the expression level of at least 1 gene or protein in the group consisting of the treated MSC is higher than that in the untreated MSC and/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is lower in the treated MSC than that in the untreated MSC.

Further, a further aspect of the present invention relates to a method of determining the suitability of an MSC for a treatment for improving the therapeutic effect of the MSC, comprising: determining the concentration of selected from p16in the treated MSCs^ink4a、p14^ARFA step of measuring the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD 47; a comparison step of comparing the measured expression level with an expression level in an untreated MSC; and is selected from the group consisting of p16^ink4aAnd p14^ARFAnd/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is greater or equal in the treated MSCs than in the untreated MSCs, the MSC is judged to be unsuitable for the exclusion determination step of the treatment.

In the adaptability judging method of the above-described aspect, the "treated MSC" means an MSC that has received some treatment for improving the therapeutic effect of the MSC. Further, "untreated MSC" means MSC that did not receive the treatment or MSC that received the treatment as a control. Examples of such treatments and their control treatments include culture in the presence/absence of an activating agent, culture on a three-dimensional culture carrier/culture on a two-dimensional culture carrier, culture under low oxygen concentration or low nutrient/culture under normal oxygen concentration or normal nutrient, culture in a microgravity environment or hypergravity environment/culture in a normal gravity environment, growth factors such as bFGF, TNF- α, IL-6, TGF- β, and PDGF, culture in the presence/absence of cytokines-chemokines, and the like, and transformation/non-treatment by gene transfer.

As described above, p16^ink4aAnd p14^ARFSince CDK4, CDK6, RB and CD47 are negatively correlated with the therapeutic effect of MSC, the MSC can be judged whether it is responsive to the treatment or adaptable to the treatment by comparing their expression levels in the treated MSC and the untreated MSC, which are used to improve the therapeutic effect of MSC.

The measurement step and the comparison step in the adaptability judging method of the above-described embodiment are described in the corresponding steps of the therapeutic effect judging method. In addition, the gene or protein expression level of the therapeutic effect marker in the untreated MSCs may be measured simultaneously with the treated MSCs or may be prepared in advance.

Selected from the group consisting of p16in processed MSCs^ink4aAnd p14^ARFIn the case where the expression amount of at least 1 gene or protein of the group consisting of more in treated MSCs than in untreated MSCs and/or the expression amount of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is less in treated MSCs than in untreated MSCs, the MSCs are predicted to be MSCs whose therapeutic effect is improved by the treatment, to be MSCs suitable for the treatment, in other words, the MSCs can be judged to be adaptive to the treatment. The adaptability judging method of the above aspect may also be expressed as a method of judging an increase in the MSC therapeutic effect by a treatment for improving the MSC therapeutic effect, including: the measurement step and the comparison step; and is selected from the group consisting of p16^ink4aAnd p14^ARFAt least 1 gene or protein of the group consisting of expressed more in treated MSCs than in untreated MSCs and/or at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47And a step of judging that the therapeutic effect of the MSC is increased by the treatment when the expression level of the proton is smaller in the treated MSC than in the untreated MSC.

In another aspect, selected from the group consisting of p16in the processed MSC^ink4aAnd p14^ARFWhen the expression level of at least 1 gene or protein of the group consisting of MSC is less than or equal to that of the untreated MSC in the treated MSC and/or when the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is more than or equal to that of the untreated MSC in the treated MSC than in the untreated MSC, it is predicted that the MSC has a low possibility of improving the therapeutic effect even if the treatment for improving the therapeutic effect is performed, and it is determined that the MSC has low suitability or no suitability for the treatment. The adaptability judging method in the above-described manner can also be expressed as a method of judging that there is no improvement in the MSC treatment effect due to the treatment for improving the MSC treatment effect, and includes: the measurement step and the comparison step; and is selected from the group consisting of p16^ink4aAnd p14^ARFAnd (b) determining that the therapeutic effect of the MSC is not improved by the treatment when the expression level of at least 1 gene or protein of the group consisting of the genes is less than or equal to that of the untreated MSC in the treated MSC and/or the expression level of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB and CD47 is more than or equal to that of the untreated MSC in the treated MSC.

In the method for determining suitability of the above-described embodiment, similarly to the method for determining therapeutic effect, clustering analysis may be used in determining suitability using a plurality of therapeutic effect markers, and the determination may be made in combination with stem cell-related factors, immunoregulatory-anti-inflammatory function-related factors, and telomerase activity-related factors, and the proportion of CD 47-positive cells in MSCs may be used instead of the expression level of CD47 gene or protein, as described in detail in the method for determining therapeutic effect.

Alternatively, instead of the treatment for improving the therapeutic effect of MSCs selected from the group consisting of p16, in the method for judging suitability in the above-described manner, a treatment for making it unclear whether or not the therapeutic effect of MSCs can be improved may be performed^ink4aAnd p14^ARFThe treatment is judged to be suitable for improving the MSC therapeutic effect in the case where the expression amount of at least 1 gene or protein of the group consisting of more in the treated MSCs than in the untreated MSCs and/or the expression amount of at least 1 gene or protein selected from the group consisting of CDK4, CDK6, RB, and CD47 is less in the treated MSCs than in the untreated MSCs. The present invention also provides such a judgment method.

The MSC that has been judged to have a good therapeutic effect, high adaptability to treatment for improving the therapeutic effect, or an improved therapeutic effect by the therapeutic effect judgment method and the adaptability judgment method can be further studied for the production of the MSC for transplantation that utilizes the treatment, or the study thereof. On the other hand, with respect to MSCs that have been judged to have poor therapeutic effects, low or no adaptability, or no improvement in therapeutic effects, the production of MSCs for transplantation by this treatment is not performed, and the consumption of MSCs and reagents such as activators necessary for the treatment can be reduced.

Cell preparation

Another embodiment of the present invention relates to a cell preparation containing MSCs, wherein the proportion of CD 47-positive cells is 2% or less.

As described above, CD47 was confirmed to be a therapeutic effect marker negatively correlated with the therapeutic effect of MSCs. The present inventors have found that MSCs with a very low proportion of CD 47-positive cells can be produced by the production method of the first embodiment. Since this MSC can be expected to have a very high therapeutic effect, a cell preparation containing the MSC can be used as a drug for treating and/or preventing diseases in which a cell transplantation therapy using MSC is effective.

The cell preparation can be produced using the MSC produced by the production method of the first embodiment. The MSC contained in the cell preparation is characterized in that the percentage of CD 47-positive cells is 2% or less. As shown in the following examples, even in MSCs collected from young people, which are considered to have a good therapeutic effect, the proportion of CD 47-positive cells exceeds 2%, and in this regard, MSCs with an extremely low proportion of CD 47-positive cells are expected to have a good therapeutic effect which has not been achieved before.

The proportion of CD 47-positive cells in the MSCs contained in the cell preparation is 2% or less, preferably 1.5% or less. The proportion of CD 47-positive cells in MSCs can be confirmed by the method mentioned in the aforementioned method for judging therapeutic effects.

The cell preparation can also be produced by concentrating MSC negative for CD47 from MSC clusters such as MSC isolated from a subject. Accordingly, the present invention also provides a method for producing a cell preparation containing MSCs, comprising a step of concentrating MSCs that are negative for CD47 from a MSC cluster.

Concentration of CD47 negative MSCs can be performed using well known techniques used in the concentration of cells that do not have specific cell surface markers, such as negative selection for CD 47. In the negative selection of CD47, MSC clusters were supplied to beads to which a CD47 specific antibody was bound, and a flow cytometer using a CD47 specific antibody, and MSCs that did not bind to the antibody were recovered.

The cell preparation contains an effective amount of MSCs. By "effective amount" is meant an amount effective for the treatment and/or prevention of disease. The effective amount is suitably adjusted depending on the kind of disease, severity of symptoms, administration route, and other medical factors of the patient. In a preferred embodiment, the effective amount of MSC is 10 per 1kg of body weight of the subject administered in the case of systemic administration⁴Cell-10⁹Cells, preferably 10⁵Cell-10⁸Cells, in the case of topical administration, 10 body weight per 1kg of the individual administered²Cell-10⁹Cells, preferably 10⁴Cell-10⁶A cell. As described above, the MSCs produced by the production method according to the first aspect of the present invention have a better therapeutic effect than MSCs activated by an activating agent on a culture carrier not having a three-dimensional structure composed of fibers, typically on a two-dimensional culture carrier, and therefore the effective amount of MSCs in a cell preparation can be 1/20 to 1/2, preferably 1/10 to 1/4, which is an effective amount of activated MSCs produced by a cell culture carrier not having a three-dimensional structure composed of fibers. The effective amount of the cell preparation may be administered 1 time or in multiple divided doses.

In addition, it is known that cultures of MSCs contain various humoral factors produced by MSCs, and still have therapeutic effects on diseases. Therefore, the culture obtained by culturing the MSCs contained in the cell preparation of the above-described manner can also be used as a drug for the treatment and/or prevention of diseases. The culture is preferably a culture supernatant, and can be obtained by culturing MSCs or activated MSCs in the presence of an activator in a medium generally used for culturing MSCs, for example, α -MEM, DMEM, or the like. The effective amount of the culture in the medicament containing the MSC culture is 0.01mg to 100mg, preferably 0.02mg to 50mg, more preferably 0.05mg to 20mg per 1kg of the body weight of the subject to be administered, and it may be administered 1 time or a plurality of times.

The cell preparation and the drug containing a culture (hereinafter collectively referred to as the drug of the present invention) are generally used in the form of a non-oral preparation such as an injection. Examples of carriers that can be used in the non-oral preparation include aqueous carriers such as physiological saline and isotonic solutions containing glucose, D-sorbitol, and the like. In addition, the drug of the present invention may be a pharmaceutical composition containing a medically acceptable buffer, stabilizer, preservative, and other ingredients.

The method of administration of the drug of the present invention is not particularly limited, and in the case of a non-oral preparation, for example, local administration (coating means coating of membrane tissue of various organs) such as intravascular administration (preferably intravenous administration), intraperitoneal administration, enteral administration, subcutaneous administration, subcapsular administration, and the like can be mentioned. In a preferred embodiment, the medicament of the invention is administered to an organism by intravenous administration. In addition, the drug of the present invention may be used in combination with other drugs depending on the diseases to be treated and/or prevented.

The cell preparation may be administered locally to the living body by adhering the cell preparation to a site where a disease occurs in the living body, a site where a disease may occur, a site which causes a disease, or the vicinity thereof in the form of adhering to a three-dimensional culture carrier. In the present embodiment, the material of the fiber forming the three-dimensional culture carrier is preferably a biocompatible material, and particularly preferably a biodegradable polymer.

The medicament of the present invention can be used for the treatment and/or prevention of diseases for which cell transplantation therapy using MSC is effective, such as diabetes and its complications, cerebrovascular diseases, brain degenerative diseases, demyelinating diseases, functional paroxysmal diseases, dementing diseases, peripheral nerve diseases, cardiovascular diseases, autoimmune diseases, liver/biliary tract/pancreas diseases, stomach/duodenal tract diseases, small/large intestine diseases, thyroid diseases, blood/hematopoietic organ diseases, lung diseases, acute renal dysfunction and chronic kidney diseases, eye diseases, skin diseases, muscle/bone diseases, trauma and GVHD (graft versus host disease). As these diseases, specifically, there can be mentioned: type I diabetes and type II diabetes and their complications such as diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, diabetic gangrene and the like; cerebrovascular diseases such as apoplexy (cerebral infarction and cerebral hemorrhage), and brain degenerative diseases such as Parkinson disease, Huntington disease, cerebral basal ganglia degeneration, multiple system atrophy, spinocerebellar degeneration, and amyotrophic lateral sclerosis; demyelinating diseases such as multiple sclerosis, acute disseminated encephalomyelitis, and neuromyelitis optica; functional paroxysmal diseases such as epilepsy and cerebral palsy; dementia diseases such as vascular cognitive disorder, Alzheimer's disease, Lewy body type cognitive disorder, cephalic-cephalic type cognitive disorder, and diabetic cognitive disorder; peripheral nerve diseases such as Guillain-Barre syndrome, peripheral nerve disorder, facial paralysis, trigeminal neuralgia, dysuria, erectile dysfunction, and autonomic nerve dysfunction; cardiovascular diseases such as myocardial infarction, stenocardia, infarct arteriosclerosis, and cardiomyopathy; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, mixed connective tissue disease, polymyalgia rheumatica, eosinophilia, Behcet's disease, sarcoidosis, Still disease, spondyloarthritis, Kawasaki disease, etc.; liver-biliary tract-pancreas diseases such as acute and chronic hepatitis, liver cirrhosis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, acute and chronic pancreatitis, and autoimmune pancreatitis; gastroduodenal diseases such as acute and chronic gastritis, gastric and duodenal ulcers; small intestine-large intestine diseases such as Crohn's disease, ulcerative colitis, ischemic colitis, and irritable bowel syndrome; thyroid diseases such as Graves' disease, acute and chronic thyroiditis; blood-hematopoietic organ diseases such as autoimmune hemolytic anemia, polycythemia vera, and idiopathic thrombocytopenic purpura; lung diseases such as chronic obstructive pulmonary disease, interstitial pneumonia, pulmonary fibrosis, pneumoconiosis, bronchial asthma, eosinophilic pneumonia, and ARDS (acute respiratory distress syndrome); acute renal dysfunction accompanying reduction of body fluid volume, acute renal dysfunction due to ischemia, ANCA-related nephritis, microscopic polyangiitis, polyangitic inflammatory granulomatosis, eosinophilic polyangitic inflammatory granulomatosis, malignant hypertension, cryoglobulinemia, postinfection glomerulonephritis, IgA nephropathy, acute interstitial nephritis, drug-induced renal dysfunction, myeloma kidney, gout kidney, rhabdomyolysis, acute tubular necrosis, and other acute renal dysfunction; chronic kidney diseases such as membranous nephropathy, membranous proliferative glomerulonephritis, minimal change nephrotic syndrome, focal glomerulosclerosis, lupus nephritis, amyloidosis, nephrosclerosis, purpuric nephritis, IgG 4-associated nephropathy, sjogren's syndrome, scleroderma kidney, chronic interstitial nephritis, and multiple renal cysts; eye diseases such as macular degeneration, optic neuritis, uveitis, etc.; atopic dermatitis, vesicular disease, Stevens-Johnson syndrome, and other skin diseases; myasthenia gravis, muscular dystrophy, osteoarthritis, femoral head necrosis, osteoporosis, carpal tunnel syndrome, and other muscular-skeletal diseases; trauma such as spinal cord injury and cerebral contusion; wound such as bedsore, oral ulcer, GVHD (graft versus host disease), anastomotic fistula, and organ injury. Preferred diseases for which the medicament of the present invention is suitable are diabetic nephropathy, acute renal dysfunction, chronic kidney disease, diabetic retinopathy, diabetic neuropathy, diabetic gangrene, alzheimer's disease, diabetic cognitive disorder, rheumatoid arthritis, polymyositis and wounds.

The treatment and/or prevention as used in the present specification includes all types of medically allowable therapeutic and/or prophylactic interventions for the purpose of cure, temporary relief, prevention, etc. of a disease or a symptom. That is, the treatment and/or prevention of a disease or symptom includes medically allowable interventions for various purposes, including delaying or stopping progression of a disease or symptom, regression or disappearance of a lesion, prevention of onset or prevention of recurrence, and the like.

The present invention also encompasses a method of administering an effective amount of the present drug to a subject in need thereof, and a method of treating and/or preventing a disease in which a cell transplantation therapy using MSCs is effective, as another mode. The terms in this embodiment have the meanings described above.

The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to these examples.