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Efficient method for nuclear reprogramming

a nuclear reprogramming and efficient technology, applied in the direction of artificial cell constructs, non-embryonic pluripotent stem cells, genetically modified cells, etc., can solve the problems of low production efficiency low efficiency, and small number of induced pluripotent stem cells, etc., to achieve efficient preparation, efficient preparation, and efficient preparation

Inactive Publication Date: 2010-03-25
KYOTO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for efficiently preparing induced pluripotent stem cells using a combination of a nuclear reprogramming factor and at least one miRNA. The methods involve introducing the nuclear reprogramming factor and miRNA into somatic cells, which results in increased efficiency of nuclear reprogramming compared to using the factor alone. The miRNA can be introduced as a pre-miRNA or as a primary miRNA. The methods can also involve the use of a retroviral vector or a vector containing the gene for the nuclear reprogramming factor. The miRNA can regulate DNA methylation or de novo DNA methylation. The methods can also involve the use of specific miRNAs or miRNA clusters. Overall, the invention provides efficient and effective methods for preparing induced pluripotent stem cells.

Problems solved by technology

However, transplantation of ES cells has a problem of causing rejection in the same manner as organ transplantation.
Moreover, from an ethical viewpoint, there are many dissenting opinions against the use of ES cells which are established by destroying human embryos.
However, all previously reported nuclear reprogramming methods mediated by the introduction of genes involve a problem of low efficiency in which only a small number of induced pluripotent stem cells can be obtained.
In particular, there is a problem in that, if reprogramming is carried out in somatic cells through the introduction of three factors (namely, Oct3 / 4, Sox2, and Klf4) excluding c-Myc, then the production efficiency of induced pluripotent stem cells becomes low.
However, these documents do not disclose any role of small RNA in the nuclear reprogramming of somatic cells.

Method used

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  • Efficient method for nuclear reprogramming
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Examples

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example 1

Preparation of Induced Pluripotent Stem Cells Through Nuclear Reprogramming of Mouse Embryonic Fibroblasts

[0107]pMXs-based retroviral vectors, which respectively encode each of three genes of mouse-derived Oct3 / 4, Sox2, and Klf4, control DsRed or each miRNA of 18 types of miRNAs, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche) to get retroviruses. On the next day, embryonic fibroblasts (Nanog GFP MEF, WO2007 / 069666 A1) derived from transgenic mice generated by insertion of sequences encoding EGFP gene and puromycin resistance gene downstream of a Nanog gene promoter region, were seeded at 1×105 cells / well in 6-well plates. On the next day, these cells were infected with retroviruses expressing Oct3 / 4, Sox2, Klf4, and each type of miRNA selected from 18 types of miRNAs, at a ratio of 1 ml of virus mixture expressing these three factors to 1 ml of virus solution expressing miRNA or DsRed, so as to prepare induced pluripotent stem cells through nuclear reprogramming.

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example 2

Preparation of Induced Pluripotent Stem Cells Through Nuclear Reprogramming of Mouse Tail Tip Fibroblasts

[0109]pMXs-based retroviral vectors, which respectively encode each of three genes of mouse-derived Oct3 / 4, Sox2, and Klf4, DsRed (control), or mmu-miR-295, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche) to get retroviruses. On the next day, tail tip fibroblasts (Nanog GFP tailtip fibroblasts) derived from transgenic mice generated by insertion of sequences encoding EGFP gene and puromycin resistance gene downstream of a Nanog gene promoter region, were seeded at 1×105 cells / well in 6-well plates. On the next day, these cells were infected with retroviruses expressing three factors of Oct3 / 4, Sox2, and Klf4, and either DsRed or mmu-miR-295, at a ratio of 1:1:1:1, so as to prepare induced pluripotent stem cells through nuclear reprogramming.

[0110]Since the third day after infection, the cells were cultured in an ES cell medium containing LIF. On the fourth day a...

example 3

Preparation of Induced Pluripotent Stem Cells Through Nuclear Reprogramming of Adult Human Dermal Fibroblasts

[0111]pMXs-based retroviral vectors, which encode three genes of human-derived OCT3 / 4, SOX2, and KLF4, and control DsRed or either 23 types of miRNAs or an miRNA cluster, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche) to get retroviruses. On the next day, adult human dermal fibroblasts (aHDF) which were generated to express a rodent ecotropic virus receptor Slc7a1 (aHDF-Slc7a1), were seeded at 3×105 cells / well in 6-cm dishes. On the next day, the cells were infected with retroviruses expressing three genes of OCT3 / 4, SOX2, KLF4, and various types of miRNAs, at a ratio of 1:1:1:1, so as to produce induced pluripotent stem cells through nuclear reprogramming.

TABLE 2miRNA sequence (othermiRNAname(s) indicated inmiRBasenumberparentheses)accession number1hsa-miR-371MI0000779(hsa-miR-371-5p / 371-3p)2hsa-miR-372MI00007803hsa-miR-373MI0000781(hsa-miR-373 / 373*)4hsa-m...

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Abstract

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.

Description

PRIOR RELATED APPLICATIONS[0001]This application is a continuation-in-part of PCT / JP2008 / 59586, filed May 23, 2008, which claims priority of U.S. Provisional Application No. 60 / 996,893, filed Dec. 10, 2007, and this application also claims priority of U.S. Provisional Application No. 60 / 996,893, filed Dec. 10, 2007. The entire disclosures of each of the above-cited applications are considered as being part of this application and are expressly incorporated by reference herein in their entireties.FIELD OF THE INVENTION[0002]The present invention relates to efficient methods for preparing induced pluripotent stem cells through reprogramming of somatic cells, to induced pluripotent stem cells, to uses of induced pluripotent stem cells and to somatic cells derived by inducing differentiation of said pluripotent cells. The present invention also relates to nuclear reprogramming factors and to miRNAs involved in generating induced pluripotent stem cells. The present invention also relates...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09C12N5/071
CPCC12N5/0696C12N15/113C12N2310/141C12N2330/10C12N2501/605C12N2510/00C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2501/65C12N2501/608C12N5/0607C12N5/10
Inventor YAMANAKAKOYANAGI, MICHIYO
Owner KYOTO UNIV
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