Efficient method for nuclear reprogramming

• US 20090246875A1
• Filed: 02/25/2009
• Published: 10/01/2009
• Est. Priority Date: 12/10/2007
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Yamanaka, Shinya, Koyanagi, Michiyo
• Granted Patent: US 9,683,232 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2330/10 naturally occurring
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/65 MicroRNA
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

This relates to a method of preparing induced pluripotent stem cells, comprising a nuclear reprogramming step with a nuclear reprogramming factor in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence thereof.

Claims

1. A method of preparing an induced pluripotent stem cell from a somatic cell, comprising nuclear reprogramming the somatic cell with at least one nuclear reprogramming factor and at least one miRNA, wherein the at least one miRNA increases efficiency of the nuclear reprogramming of the somatic cell compared to nuclear reprogramming of the somatic cell with the at least one nuclear reprogramming factor in the absence of the at least one miRNA, and wherein the at least one nuclear reprogramming factor comprises at least (i) an Oct family gene, (ii) an Oct family gene and a Klf family gene, (iii) an Oct family gene and a Nanog gene, or (iv) an Oct family gene, Klf family gene, and a Myc family gene, but it does not comprise a Sox family gene.

3. The method according to claim 1 or 2, wherein the at least one miRNA is expressed in embryonic stem cells at a higher level than in somatic cells.

4. The method according to claim 1 or 2, wherein the Oct family gene is an Oct3/4 gene, the Klf amily gene is a Klf4 gene, the Myc family gene is a c-Myc gene, or the Sox family gene is a Sox2 gene.

5. The method according to claim 1 or 2, wherein the at least one nuclear reprogramming factor is (i) an Oct3/4 gene, (ii) a combination of an Oct3/4 gene and a Klf4 gene, (iii) a combination of an Oct3/4 gene and a Nonog gene, or (iv) a combination of an Oct3/4 gene, a Klf4 gene and a c-Myc gene.

6. The method according to claim 1 or 2, wherein the at least one nuclear reprogramming factor is introduced in the form of a vector comprising DNA encoding the nuclear reprogramming factor, into the somatic cell.

7. The method according to claim 1 or 2, wherein the at least one miRNA is introduced in the form of a vector comprising DNA encoding the miRNA or a precursor RNA thereof, into the somatic cell.

8. The method according to claim 1 or 2, wherein the at least one miRNA is one or two or more miRNAs contained in one or two or more RNAs selected from the group consisting of:

hsa-miR-372;

hsa-miR-373 or hsa-miR-373/373*;

hsa-miR-371-373 cluster;

hsa-miR-302b or hsa-miR-302b/302b*;

hsa-miR-302-367 cluster;

hsa-miR-520c or hsa-miR-520c-5p/520c-3p;

mmu-miR-291a or mmu-miR-291a-5p/291a-3p;

mmu-miR-294 or mmu-miR-294/294*; and

mmu-miR-295 or mmu-miR-295/295*”

, where the symbols represent accession numbers from the miRBase database.

9. The method according to claim 1 or 2, wherein the at least one miRNA is one or two or more miRNAs contained in one or two or more RNAs selected from the group consisitng of RNAs represented by SEQ ID NOS:

1 to 5, 10 to 12, and 14.

10. The method according to claim 1 or 2, wherein the at least one miRNA is 18-25 nucleotides in length.

11. The method according to claim 1 or 2, wherein the somatic cell is of a human.

2. A method of increasing the efficiency of nuclear reprogramming for inducing an induced pluripotent stem cell from a somatic cell, comprising subjecting the somatic cell to nuclear reprogramming with at least one nuclear reprogramming factor in the presence of at least one miRNA, so that the number of induced pluripotent stem cells produced is greater than in the absence of the miRNA, wherein the at least one miRNA increases efficiency of the nuclear reprogramming of the somatic cell compared to nuclear reprogramming of the somatic cell with the at least one nuclear reprogramming factor in the absence of the at least one miRNA, and wherein the at least one nuclear reprogramming factor comprises at least (i) an Oct family gene, (ii) an Oct family gene and a Klf family gene, (iii) an Oct family gene and a Nanog gene, or (iv) an Oct family gene, Klf family gene, and a Myc family gene, but it does not comprise a Sox family gene