Method for evaluating state of differentiation of cells, method for determining state of differentiation of cells, method for producing cardiac muscle cells, and kit

Abstract

The purpose of the present invention is to provide a means for making it possible to evaluate the state of differentiation of cells with higher accuracy. By providing a first induction treatment for inducing the differentiation of pluripotent stem cells into mesoderm cells and a second induction treatment for inducing the differentiation of mesoderm cells into cardiac muscle cells, the differentiation of pluripotent stem cells into cardiac muscle cells is induced in a liquid culture medium, and a supernatant of the liquid culture medium containing the cells differentiated and induced by the second induction treatment is collected. The purpose of the present invention is to provide a method for determining the differentiation state of a cell on the basis of a measured miRNA-3p value by measuring the miRNA-3p of the miR-1 / 133a cluster in the supernatant, and a method for determining the differentiation state of the cell on the basis of the measured miRNA-3p value.

Description

[Technical field] The present invention relates to methods for evaluating the differentiation state of cells. The present invention relates to methods for determining the differentiation state of cells. The present invention relates to methods for producing cardiomyocytes. The present invention provides kits for use in these methods. [Background technology] In recent years, as an alternative treatment for heart transplantation, regenerative medicine using cardiomyocytes obtained by differentiation induction of pluripotent stem cells has attracted attention. However, it is often difficult to obtain cardiomyocytes in large quantities with high efficiency by differentiation induction. For example, due to the influence of differences in the quality of the pluripotent stem cells used, there is a deviation in the differentiation efficiency to cardiomyocytes. In order to apply the cardiomyocytes obtained by differentiation induction to patients, it is necessary to evaluate the differentiation state of the cells. In the evaluation of the differentiation state from pluripotent stem cells to cardiomyocytes, cardiac troponin T (cTnT) as a marker of cardiomyocytes has been used in the past. In this evaluation method, first, a portion of cells after differentiation induction are collected and immunostained with fluorescently labeled anti-cTnT antibodies. Furthermore, cells expressing cTnT are detected by flow cytometry, and the proportion of cells expressing cTnT (cTnT positive rate) is calculated among the cells after differentiation induction. In the past, the differentiation state to cardiomyocytes was evaluated based on this cTnT positive rate. However, since fluorescently labeled cells cannot be used in transplantation to a living body, this evaluation method can be called a destruction inspection. Since pluripotent stem cells and cells prepared therefrom are expensive, it is not good to consume a part of them for quality evaluation. On the one hand, as an example of non-destructive inspection, Non-Patent Document 1 discloses that the differentiation state of the cells is evaluated by measuring the amount of miRNA in the culture supernatant during the differentiation induction process from human embryonic stem cells to cardiomyocytes. [Prior art literature] [Patent Literature] [Non-patent document 1] Zhang Y. et al., A non-invasive method to determine the pluripotent status of stem cells by culture medium microRNA expression detection (2016) Scientific Reports, vol.6: 22380 [Summary of the Invention] [Problems to be solved by the invention] In non-patent literature 1, in order to evaluate the differentiation state of human embryonic stem cells to cardiomyocytes, miRNAs known to be expressed only in mice or miRNAs known to be expressed in small amounts are also measured. Therefore, the accuracy of the evaluation of the differentiation state is in doubt. The present invention aims to provide a means to enable the differentiation state of cells to be evaluated with higher accuracy.

Methods for solving the problem

[24] are provided.

[0001] A method for evaluating the differentiation state of cells, comprising: a first induction treatment for inducing differentiation of pluripotent stem cells into mesoderm cells and a second induction treatment for inducing differentiation of the mesoderm cells into cardiomyocytes, a step of inducing differentiation of pluripotent stem cells into cardiomyocytes in a liquid culture medium, a step of collecting the supernatant of the liquid culture medium containing cells induced to differentiate by the second induction treatment, and a step of measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant, wherein the miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p, and the measured value of the miRNA-3p serves as an indicator of differentiation into cardiomyocytes.

[0002] [1] The method according to claim 1, wherein the miRNA-3p is the miR-1-3p, and when the measured value of the miR-1-3p is less than a first threshold value, it is indicated that the differentiation-induced cells are not suitable for transplantation.

[0003] [1] The method according to claim 1, wherein the miRNA-3p is miR-133a-3p, and when the measured value of the miR-133a-3p is less than a second threshold value, it is indicated that the differentiation-induced cells are not suitable for transplantation.

[0004] [1] The method described above, wherein when the miRNA-3p is the miR-1-3p and the miR-133a-3p, and the measured value of the miR-1-3p is less than a first threshold value, and the measured value of the miR-133a-3p is less than a second threshold value, it is indicated that the differentiation-induced cells are not suitable for transplantation.

[0005] [1] The method described above, wherein when the miRNA-3p is the miR-1-3p and the miR-133a-3p, and the measured value of the miR-1-3p is less than the first threshold value, or the measured value of the miR-133a-3p is less than the second threshold value, it is indicated that the differentiation-induced cells are not suitable for transplantation.

[0006] The method according to any one of [1] to [5], wherein the first induction treatment uses a liquid culture medium containing at least one of a substance promoting a BMP signaling system and a substance promoting a Wnt signaling system.

[0007] The method according to any one of [1] to [6], wherein the second induction treatment uses a liquid culture medium containing a substance that inhibits the Wnt signal transduction system.

[0008] The method described in any one of [1] to [7] further includes a step of inducing differentiation by the first induction treatment, collecting the supernatant of the liquid culture medium containing the cells before the second induction treatment, and measuring miR-489-3p in the supernatant, wherein the measured value of miR-489-3p serves as an indicator of differentiation into mesoderm cells.

[0009] [8] The method according to claim 1, wherein when the measured value of the miR-489-3p is above a third threshold value, it indicates that the pluripotent stem cells differentiate into mesodermal cells.

[0010] A method for determining the differentiation state of cells, comprising: a differentiation induction step comprising a first induction treatment for inducing differentiation of pluripotent stem cells into mesoderm cells in a liquid culture medium and a second induction treatment for inducing differentiation of the mesoderm cells into cardiomyocytes, a step of collecting the supernatant of the liquid culture medium containing the cells induced to differentiate by the second induction treatment, a step of measuring the miRNA-3p of the miR-1 / 133a cluster in the supernatant, and a step of determining that the differentiation-induced cells are not suitable for transplantation when the measured value of the miRNA-3p is less than the threshold value, wherein the miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p.

[0011]

[10] The method described above, wherein the miRNA-3p is the miR-1-3p, the threshold is the first threshold, and in the determination step, when the measured value of the miR-1-3p is less than the first threshold, the differentiation-induced cells are determined to be unsuitable for transplantation.

[0012]

[10] The method described above, wherein the miRNA-3p is the miR-133a-3p, the threshold is a second threshold, and in the determination step, when the measured value of the miR-133a-3p is less than the second threshold, the differentiation-induced cells are determined to be unsuitable for transplantation.

[0013]

[10] The method described above, wherein the miRNA-3p is the miR-1-3p and the miR-133a-3p, the threshold is the first threshold and the second threshold, and in the judgment step, when the measured value of the miR-1-3p is less than the first threshold and the measured value of the miR-133a-3p is less than the second threshold, it is judged that the differentiation-induced cells are not suitable for transplantation.

[0014]

[10] The method described above, wherein the miRNA-3p is the miR-1-3p and the miR-133a-3p, the threshold is the first threshold and the second threshold, and in the judgment step, when the measured value of the miR-1-3p is less than the first threshold, or the measured value of the miR-133a-3p is less than the second threshold, it is judged that the differentiation-induced cells are not suitable for transplantation.

[0015] The method according to any one of

[10] to

[14] , comprising the step of performing the first induction treatment using a liquid culture medium containing at least one of a substance promoting a BMP signaling system and a substance promoting a Wnt signaling system.

[0016] The method according to any one of

[10] to

[15] , wherein the second induction treatment uses a liquid culture medium containing a substance that inhibits the Wnt signal transduction system.

[0017] The method described in any one of

[10] to

[16] further includes: a step of inducing differentiation by the above-mentioned first induction treatment, collecting the supernatant of the liquid culture medium containing the cells before the above-mentioned second induction treatment, measuring the miR-489-3p in the above-mentioned supernatant, and a step of determining that the above-mentioned pluripotent stem cells have differentiated into mesoderm cells when the measured value of the above-mentioned miR-489-3p is above the above-mentioned third threshold value.

[0018] The method described in any one of

[10] to

[17] further includes: a step of inducing differentiation by the first induction treatment, collecting the supernatant of the liquid culture medium containing the cells before the second induction treatment, measuring the miR-489-3p in the supernatant, and determining that the differentiation of the pluripotent stem cells into mesoderm cells is insufficient when the measured value of the miR-489-3p is less than a third threshold value.

[0019] A method for producing cardiomyocytes, comprising: (1) a step of inducing the differentiation of mesodermal cells into cardiomyocytes in a liquid culture medium by inducing the differentiation of mesodermal cells into cardiomyocytes; (2) a step of collecting the supernatant of the liquid culture medium containing the cells induced in the above step (1); (3) a step of measuring the miRNA-3p of the miR-1 / 133a cluster in the supernatant collected in the above step (2); and (4) when the measured value of miRNA-3p is above the above threshold value, a step of culturing the cells induced to differentiate in the above step (1) to obtain cardiomyocytes, wherein the above miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p.

[0020]

[19] The method according to claim 1, wherein in the step (4), when the measured value of the miRNA-3p is less than a threshold value, the culture of the cells induced to differentiate in the step (1) is terminated.

[0021] The production method described in

[19] or

[20] performs: (i) a step of inducing the differentiation of pluripotent stem cells into mesoderm cells in a liquid culture medium by inducing the differentiation of pluripotent stem cells into mesoderm cells, (ii) a step of collecting the supernatant of the liquid culture medium containing the cells induced in the above step (i), and (iii) a step of measuring miR-489-3p in the supernatant collected in the above step (ii), and when the measured value of the above miR-489-3p is above a third threshold value, the cells in the above step (ii) are used as the mesoderm cells in the above step (1).

[0022]

[21] The method according to claim 1, wherein when the measured value of the miR-489-3p is less than a threshold value, the culturing of the cells in the step (ii) is terminated.

[0023] A kit for use in the method described in any one of [1] to [7],

[10] to

[16] ,

[19] and

[20] , comprising oligonucleotide primers and / or oligonucleotide probes for detecting miRNA-3p of the miR-1 / 133a cluster.

[0024] A kit for use in the method described in any one of [8], [9],

[17] ,

[18] ,

[21] and

[22] , comprising oligonucleotide primers and / or oligonucleotide probes for detecting miRNA-3p of the miR-1 / 133a cluster and oligonucleotide primers and / or oligonucleotide probes for detecting miR-489-3p. [Effects of the invention] The present invention makes it possible to evaluate the differentiation state of cells with high accuracy. [Brief description of the attached drawings]

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Table 1

Table 2

Explanation of symbols

Claims

1. A method for evaluating the differentiation state of a cell, comprising: A process for inducing pluripotent stem cells to differentiate into cardiomyocytes in a liquid culture medium by the following induction treatments: a first induction treatment for inducing differentiation of pluripotent stem cells into mesoderm cells, and a second induction treatment for inducing differentiation of the mesodermal cells into cardiomyocytes, a step of collecting the supernatant of the liquid culture medium containing cells induced to differentiate by the second induction treatment, and a step of measuring miRNA-3p clustered with miR-1 / 133a in the supernatant, The miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p, The measured value of miRNA-3p serves as an indicator of differentiation into cardiomyocytes.

2. The method of claim 1, wherein The miRNA-3p is the miR-1-3p, When the measured value of the miR-1-3p is less than the first threshold value, it is suggested that the differentiation-induced cells are not suitable for transplantation.

3. The method of claim 1, wherein The miRNA-3p is the miR-133a-3p, When the measured value of the miR-133a-3p is less than the second threshold value, it is suggested that the differentiation-induced cells are not suitable for transplantation.

4. The method of claim 1, wherein The miRNA-3p is the miR-1-3p and the miR-133a-3p, When the measured value of the miR-1-3p is less than the first threshold value and the measured value of the miR-133a-3p is less than the second threshold value, it is suggested that the differentiation-induced cells are not suitable for transplantation.

5. The method of claim 1, wherein The miRNA-3p is the miR-1-3p and the miR-133a-3p, When the measured value of the miR-1-3p is less than the first threshold value, or when the measured value of the miR-133a-3p is less than the second threshold value, it is suggested that the differentiation-induced cells are not suitable for transplantation. 6 . The method according to claim 1 , wherein the first induction treatment comprises using a liquid culture medium containing at least one of a substance that promotes a BMP signaling system and a substance that promotes a Wnt signaling system.

7. The method of claim 1, wherein the second induction treatment comprises using a liquid culture medium containing a substance that inhibits the Wnt signaling system.

8. The method according to claim 1, further comprising the step of collecting a supernatant of a liquid culture medium containing cells that have been induced to differentiate by the first induction treatment and before being subjected to the second induction treatment, and measuring miR-489-3p in the supernatant, The measured value of miR-489-3p serves as an indicator of differentiation into mesodermal cells. 9 . The method according to claim 8 , wherein when the measured value of the miR-489-3p is above a third threshold value, it indicates that the pluripotent stem cells differentiate into mesoderm cells.

10. A method for determining the differentiation state of a cell, comprising: Differentiation induction process in liquid culture medium: it includes: a first induction treatment for inducing differentiation of pluripotent stem cells into mesoderm cells, and a second induction treatment for inducing differentiation of the mesodermal cells into cardiomyocytes, a step of collecting the supernatant of the liquid culture medium containing the cells induced to differentiate by the second induction treatment, a step of measuring miRNA-3p clustered with miR-1 / 133a in the supernatant, and a step of determining that the differentiation-induced cells are not suitable for transplantation when the measured value of the miRNA-3p is less than a threshold value; The miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p.

11. The method of claim 10, wherein The miRNA-3p is the miR-1-3p, The threshold is the first threshold, In the determination step, when the measured value of the miR-1-3p is less than the first threshold value, it is determined that the differentiation-induced cells are not suitable for transplantation.

12. The method of claim 10, wherein The miRNA-3p is the miR-133a-3p, The threshold is the second threshold, In the determination step, when the measured value of the miR-133a-3p is less than the second threshold value, it is determined that the differentiation-induced cells are not suitable for transplantation.

13. The method of claim 10, wherein The miRNA-3p is the miR-1-3p and the miR-133a-3p, The thresholds are the first threshold and the second threshold, In the determination step, when the measured value of the miR-1-3p is less than the first threshold value and the measured value of the miR-133a-3p is less than the second threshold value, it is determined that the differentiation-induced cells are not suitable for transplantation.

14. The method of claim 10, wherein The miRNA-3p is the miR-1-3p and the miR-133a-3p, The thresholds are the first threshold and the second threshold, In the determination step, when the measured value of the miR-1-3p is less than the first threshold value, or the measured value of the miR-133a-3p is less than the second threshold value, it is determined that the differentiation-induced cells are not suitable for transplantation. 15 . The method according to claim 10 , wherein the first induction treatment comprises an induction treatment using a liquid culture medium containing at least one of a substance that promotes a BMP signaling system and a substance that promotes a Wnt signaling system.

16. The method of claim 10, wherein the second induction treatment comprises using a liquid culture medium containing a substance that inhibits the Wnt signaling system.

17. The method of claim 10, further comprising: a step of collecting a supernatant of a liquid culture medium containing cells that have been induced to differentiate by the first induction treatment and before being subjected to the second induction treatment, and measuring miR-489-3p in the supernatant; and When the measured value of the miR-489-3p is equal to or greater than a third threshold value, it is determined that the pluripotent stem cells are in the process of differentiating into mesoderm cells.

18. The method of claim 10, further comprising: a step of collecting a supernatant of a liquid culture medium containing cells that have been induced to differentiate by the first induction treatment and before being subjected to the second induction treatment, and measuring miR-489-3p in the supernatant; and When the measured value of the miR-489-3p is less than the third threshold value, it is determined that the differentiation of the pluripotent stem cells into mesoderm cells is insufficient.

19. A method for producing cardiomyocytes, comprising: (1) a step of inducing the differentiation of mesodermal cells into cardiomyocytes in a liquid culture medium by inducing the differentiation of mesodermal cells into cardiomyocytes; (2) a step of collecting the supernatant of the liquid culture medium containing the cells induced in the step (1); (3) a step of measuring miRNA-3p clustered with miR-1 / 133a in the supernatant collected in step (2); and (4) when the measured value of the miRNA-3p is equal to or greater than a threshold value, culturing the cells induced to differentiate in the step (1) to obtain cardiomyocytes, The miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p.

20. The method according to claim 19, wherein in the step (4), when the measured value of the miRNA-3p is less than a threshold value, the culture of the cells induced to differentiate in the step (1) is terminated.

21. The production method according to claim 19, which is carried out: (i) a step of inducing the differentiation of pluripotent stem cells into mesoderm cells in a liquid culture medium by an induction treatment for inducing the differentiation of pluripotent stem cells into mesoderm cells, (ii) collecting the supernatant of the liquid culture medium containing the cells induced in the step (i); and (iii) a step of measuring miR-489-3p in the supernatant collected in the step (ii), When the measured value of miR-489-3p is above the third threshold value, the cells of step (ii) are used as the mesoderm cells of step (1).

22. The method according to claim 21, wherein when the measured value of miR-489-3p is less than a threshold value, the culturing of the cells in step (ii) is terminated.

23. A kit for use in the method according to any one of claims 1 to 7, 10 to 16, 19 and 20, comprising oligonucleotide primers and / or oligonucleotide probes for detecting miRNA-3p of the miR-1 / 133a cluster.

24. A kit for use in the method of any one of claims 8, 9, 17, 18, 21 and 22, comprising: Oligonucleotide primers and / or oligonucleotide probes for detecting miRNA-3p of the miR-1 / 133a cluster, and Oligonucleotide primers and / or oligonucleotide probes for detecting miR-489-3p.

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