Method for sorting cardiomyocytes

Patent metadata

Field	Value
Publication number	US-10538740-B2
Application number	US-201515127134-A
Country	US
Kind code	B2
Filing date	Mar 20, 2015
Priority date	Mar 20, 2014
Publication date	Jan 21, 2020
Grant date	Jan 21, 2020

Abstract

An object of the present invention is to provide a novel method for sorting cardiomyocytes. Another object of the present invention is to provide a method for producing high-purity cardiomyocytes and a kit used therefor. The present invention provides a method for sorting cardiomyocytes, comprising a step of introducing miRNA-responsive mRNA into a cell group, wherein the miRNA-responsive mRNA consists of a sequence comprising the following (i) and (ii):
(i) a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes, and (ii) a nucleic acid corresponding to the coding region of a gene, wherein translation of (ii) the nucleic acid corresponding to the coding region of a gene into protein is regulated by the nucleic acid sequence in (i) above, thereby achieving the aforementioned objects.

First claim

The invention claimed is:

1. A method for providing cells useful for sorting cardiomyocytes, comprising steps of:
(A) introducing miRNA-responsive mRNA into a cell group, wherein the miRNA-responsive mRNA comprises a sequence having:
(a) a cap structure;
(b) a 5′ untranslated region (5′-UTR);
(c) an open reading frame comprising a nucleic acid corresponding to the coding region of a gene;
(d) a 3′ untranslated region (3′-UTR); and
(e) a poly (A) tail region,
wherein the 5′-UTR comprises a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes,
wherein the miRNA-responsive mRNA is introduced into the cell group in a form of a synthetic RNA molecule without using a DNA or virus vector, and
wherein translation of the nucleic acid corresponding to the coding region of a gene into protein is regulated by the nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes; and
(B) inhibiting translation of the coding region of the gene in response to the presence of an miRNA specifically expressed in the cardiomyocytes or performing translation of the coding region of the gene in response to the absence of an miRNA specifically expressed in the cardiomyocytes.

2. The method according to claim 1 , wherein the 3′-UTR comprises a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes.

3. The method according to claim 1 , wherein the miRNA specifically expressed in cardiomyocytes is one or more miRNAs selected from the group consisting of miR-1, miR-208a, miR-208b and miR-499a-5p.

4. The method according to claim 1 , wherein the gene in (c) is one or more genes selected from the group consisting of a gene encoding a fluorescent protein, an apoptosis-inducing gene and a suicide gene.

5. The method according to claim 4 , wherein the gene encoding a fluorescent protein is a gene encoding a blue fluorescent protein (BFP).

6. The method according to claim 4 , wherein the apoptosis-inducing gene is a gene encoding a Bim protein.

7. The method according to claim 1 , further comprising a step of introducing mRNA consisting of a sequence comprising a nucleic acid corresponding to the coding region of a drug resistance gene into the cell group.

8. The method according to claim 7 , wherein the drug resistance gene is an antibiotic resistance gene.

9. The method according to claim 8 , wherein the antibiotic resistance gene is a puromycin resistance gene or a blasticidin resistance gene.

10. The method according to claim 1 , wherein the cell group of step (A) is a cell group that has been induced to differentiate from pluripotent stem cells.

11. A method for producing cardiomyocytes, comprising the following steps:
(A) introducing miRNA-responsive mRNA into a cell group, wherein the miRNA-responsive mRNA comprises a sequence having:
(a) a cap structure;
(b) a 5′ untranslated region (5′-UTR);
(c) an open reading frame comprising a nucleic acid corresponding to the coding region of a gene;
(d) a 3′ untranslated region (3′-UTR); and
(e) a poly (A) tail region,
wherein the 5′-UTR comprises a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes, and
wherein the miRNA-responsive mRNA is introduced into the cell group in a form of a synthetic RNA molecule without using a DNA or virus vector;
(B) inhibiting translation of the coding region of the gene in response to the presence of an miRNA specifically expressed in the cardiomyocytes or performing translation of the coding region of the gene in response to the absence of an miRNA specifically expressed in the cardiomyocytes; and
(C) sorting the cells based on a translation level of a protein from the mRNA of step (A), wherein high-purity cardiomyocytes can be obtained compared with a case in which steps (A), (B), and (C) are not carried out.

12. The method according to claim 1 , further comprising before step (A), a step of inducing and differentiating pluripotent stem cells into cardiomyocytes to prepare the cell group.

13. The method according to claim 12 , wherein the inducing and differentiating step comprises the following substeps (1) to (3):
(1) culturing the pluripotent stem cells in a culture medium containing BMP, bFGF and Activin A;
(2) culturing the pluripotent stem cells in a culture medium containing VEGF and a Wnt inhibitor; and
(3) culturing the pluripotent stem cells in a culture medium containing VEGF and bFGF.

14. The method according to claim 11 , wherein the 3′-UTR comprises a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes.

15. The method according to claim 11 , wherein the miRNA specifically expressed in cardiomyocytes is one or more miRNAs selected from the group consisting of miR-1, miR-208a, miR-208b and miR-499a-5p.

16. The method according to claim 11 , wherein the gene in (c) is a gene encoding a fluorescent protein.

17. The method according to claim 16 , wherein the gene encoding a fluorescent protein is a gene encoding BFP.

18. The method according to claim 11 , wherein the cell group of step (A) is a cell group that has been induced to differentiate from pluripotent stem calls.

19. A method for producing cardiomyocytes, comprising:
(A) introducing miRNA-responsive mRNA and mRNA consisting of a sequence comprising a nucleic acid corresponding to the coding region of a drug resistance gene into a cell group, and
(B) culturing the cell group obtained in step (A) in the presence of a drug corresponding to the drug resistance gene of step (A) wherein the miRNA-responsive mRNA comprises a sequence having:
(a) a cap structure;
(b) a 5′ untranslated region (5′-UTR);
(c) an open reading frame comprising a nucleic acid corresponding to the coding region of a gene;
(d) a 3′ untranslated region (3′-UTR); and
(e) a poly (A) tail region,
wherein the 5′-UTR comprises a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes,
wherein the miRNA-responsive mRNA is introduced into the cell group in a form of a synthetic RNA molecule without using a DNA or virus vector, and
wherein high-purity cardiomyocytes can be obtained compared with a case in which steps (A) and (B) are not carried out.

20. The method according to claim 19 , wherein the miRNA-responsive mRNA is miRNA-responsive OFF switch mRNA.

21. The method according to claim 19 , wherein the 3′-UTR comprises a nucleic acid specifically recognized by miRNA specifically expressed in cardiomyocytes.

22. The method according to claim 19 , wherein the miRNA specifically expressed in cardiomyocytes is one or more miRNAs selected from the group consisting of miR-1, miR-208a, miR-208b and miR-499a-5p.

23. The method according to claim 19 , wherein the gene in (c) is an apoptosis-inducing gene and/or a suicide gene.

24. The method according to claim 23 , wherein the apoptosis-inducing gene is a gene encoding a Bim protein.

25. The method according to claim 19 , wherein the drug resistance gene is an antibiotic resistance gene.

26. The method according to claim 25 , wherein the antibiotic resistance gene is a puromycin resistance gene or a blasticidin resistance gene.

27. The method according to claim 19 , wherein the cell group of step (A) is a cell group that has been induced to differentiate from pluripotent stem cells.

28. A kit for purifyin

Assignees

• Univ Kyoto

Inventors

• Yoshida Yoshinori
• Saito Hirohide
• Miki Kenji
• Endo Kei
• Takahashi Seiya

Classifications

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• A01K2267/0375

  Animal model for cardiovascular diseases · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title