Efficient method for nuclear reprogramming

• US 9,683,232 B2
• Filed: 02/25/2009
• Issued: 06/20/2017
• Est. Priority Date: 12/10/2007
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Yamanaka, Shinya, Koyanagi, Michiyo
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US12/379,564
• Publication Number:
  US 20090246875A1
• Time in Patent Office: 3,037 Days
• 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 mammalian somatic cell, comprising nuclear reprogramming the mammalian somatic cell under culture conditions which allow for mammalian somatic cell reprogramming to pluripotency with at least one miRNA and an expression vector encoding at least one nuclear reprogramming factor,

wherein the at least one miRNA increases efficiency of the nuclear reprogramming of the mammalian somatic cell compared to nuclear reprogramming of the mammalian somatic cell with the expression vector in the absence of the at least one miRNA,

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*, and

wherein the at least one nuclear reprogramming factor comprises at least (i) an Oct3/4 gene, (ii) an Oct3/4 gene and a Klf4 gene, (iii) an Oct3/4 gene and a Nanog gene, or (iv) an Oct3/4 gene, Klf4 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 Myc family gene is a c-Myc gene.

5. The method according to claim 1 or 2, wherein the at least one nuclear reprogramming factor is an Oct3/4 gene.

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

7. 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 RNAs represented by SEQ ID NOS:

1 to 5, 10 to 12, and 14.

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

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

2. A method of increasing the efficiency of nuclear reprogramming for inducing an induced pluripotent stem cell from a mammalian somatic cell, comprising

subjecting the mammalian somatic cell to nuclear reprogramming with an expression vector encoding at least one nuclear reprogramming factor in the presence of at least one miRNA under culture conditions which allow for mammalian somatic cell reprogramming to pluripotency,

wherein the at least one miRNA increases efficiency of the nuclear reprogramming of the mammalian somatic cell compared to nuclear reprogramming of the somatic cell with the expression vector in the absence of the at least one miRNA,

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*, and

wherein the at least one nuclear reprogramming factor comprises at least (i) an Oct3/4 gene, (ii) an Oct3/4 gene and a Klf4 gene, (iii) an Oct3/4 gene and a Nanog gene, or (iv) an Oct3/4 gene, a Klf4 gene, and a Myc family gene, but it does not comprise a Sox family gene.