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Nuclear reprogramming factor and induced pluripotent stem cells

a technology of nuclear reprogramming factor and induced pluripotent stem cells, which is applied in the direction of peptides, viruses/bacteriophages, and genetically modified cells, etc., can solve the problems of human embryo use, ethical controversies, and hinder the application of human es cells

Inactive Publication Date: 2009-09-10
KYOTO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0171]By using the nuclear reprogramming factor of the present invention, the nucleus of a somatic cell can be reprogrammed to obtain an induced pluripotent stem cell. In the specification, the term “induced pluripotent stem cells” means cells having properties similar to those of ES cells, and more specifically, the term encompasses undifferentiated cells having pluripotency and growth ability. However, the term should not be construed narrowly in any sense, and should be construed in the broadest sense. The method for preparing induced pluripotent stem cells by using a nuclear reprogramming factor is explained in International Publication WO2005 / 80598 (the term “ES-like cells” is used in the publication), and a means for isolating induced pluripotent stem cells is also specifically explained. Therefore, by referring to the aforementioned publication, those skilled in the art can easily prepare induced pluripotent stem cells by using the nuclear reprogramming factor of the present invention. Methods for preparing induced pluripotent stem cells from somatic cells by using the nuclear reprogramming factor of the present invention are not particularly limited. Any method may be employed as long as the nuclear reprogramming factor can contact with somatic cells under an environment in which the somatic cells and induced pluripotent stem cells can proliferate. An advantage of the present invention is that an induced pluripotent stem cell can be prepared by contacting a nuclear reprogramming factor with a somatic cell in the absence of eggs, embryos, or embryonic stem (ES) 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.
Use of human embryos, however, faces ethical controversies that hinder the applications of human ES cells.
In addition, it is difficult to generate patient- or disease-specific ES cells, which are required for their effective application.
However, these methods all have serious drawbacks.
Methods of nuclear transfer into oocytes and techniques which involve the fusion of ES and differentiated cells both comprise the use of ES cells, which present ethical problems.
In addition, cells generated by such methods often lead to problems with rejection upon transplantation into an unmatched host.
Furthermore, the use of cell extracts to treat differentiated cells is technically unreliable and unsafe, in part because the cell extract components responsible for the nuclear programming are mixed in solution with other unknown factors.
However, this publication fails to report an actual identification of a nuclear reprogramming factor.

Method used

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  • Nuclear reprogramming factor and induced pluripotent stem cells
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example 1

Selection of a Nuclear Reprogramming Factor

[0194]In order to identify reprogramming factors, an experimental system for easy observation of the reprogramming phenomenon is required. As an experimental system, a mouse in which βgeo (a fusion gene of β-galactosidase gene and neomycin resistance gene) was knocked into the Fbx15 locus (Tokuzawa et al. Mol. Cell. Biol. 23:2699-708, 2003) was used. The mouse Fbx15 gene is a gene specifically expressed in differentiation pluripotent cells such as ES cells and early embryos. However, in a homomutant mouse in which βgeo was knocked into the mouse Fbx15 gene so as to delete the function of Fbx15, no abnormal phenotypes including those concerning differentiation pluripotency or development were observed. In this mouse, expression control of βgeo is attained by the enhancer and promoter of the Fbx15 gene. Specifically, βgeo is not expressed in differentiated somatic cells, and they have sensitivity to G418. In contrast, the βgeo knockin homomut...

example 2

Induction of Reprogramming with a Combination of 4 Kinds of Genes

[0199]It was examined whether or not induction of reprogramming of somatic cells was achievable with the four kinds of genes of which particular importance was suggested among the 10 genes. By using the combination of the aforementioned 10 kinds of genes, the combination of the aforementioned 4 kinds of genes, combinations of only 3 kinds of genes among the 4 kinds of genes, and combinations of only 2 kinds of genes among the 4 kinds of genes, these sets of genes were retrovirally transduced into the MEF cells as somatic cells in which βgeo was knocked into the Fbx15 gene. As a result, when the 4 kinds of genes were transduced, 160 G418-resistant colonies were obtained. Although this result was almost the same as that obtained by the transduction with the 10 kinds of genes (179 colonies), the colonies obtained by the 4-gene transduction were smaller than those by the 10-gene transduction. When these colonies were passa...

example 3

Analysis of Pluripotency of Reprogrammed Cells

[0204]In order to evaluate the differentiation pluripotency of the established iPS cells, the iPS cells established with 24 factors, 10 factors, and 4 factors were subcutaneously transplanted into nude mice. As a result, tumors having a size similar to that observed with ES cells were formed in all animals. Histologically, the tumors consisted of a plurality of kinds of cells, and cartilaginous tissues, nervous tissues, muscular tissues, fat tissues, and intestinal tract-like tissues were observed (FIG. 8), which verified pluripotency of the iPS cells. In contrast, although tumors were formed when the cells established with the 3 factors were transplanted into nude mice, they were formed histologically only from undifferentiated cells. These results suggested that a Sox family gene was essential for the induction of differentiation pluripotency.

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Abstract

The present invention relates to a nuclear reprogramming factor having an action of reprogramming a differentiated somatic cell to derive an induced pluripotent stem (iPS) cell. The present invention also relates to the aforementioned iPS cells, methods of generating and maintaining iPS cells, and methods of using iPS cells, including screening and testing methods as well as methods of stem cell therapy. The present invention also relates to somatic cells derived by inducing differentiation of the aforementioned iPS cells.

Description

PRIOR RELATED APPLICATIONS[0001]This application is a continuation-in-part of U.S. application Ser. No. 12 / 213,035, filed Jun. 13, 2008, which is a continuation-in-part of PCT / JP2006 / 324881, filed Dec. 6, 2006, which claims priority to Japanese Application No. 2005-359537, filed Dec. 13, 2005, and also claims priority to U.S. Provisional Application No. 61 / 001,108, filed Oct. 31, 2007, and U.S. Provisional Application No. 60 / 996,289, filed Nov. 9, 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 a nuclear reprogramming factor having an action of reprogramming a somatic cell to derive an induced pluripotent stem (iPS) cell. The present invention also relates to the aforementioned iPS cells, methods of generating and maintaining iPS cells, and methods of using iPS cells, including screen...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N5/00C12N5/08C12N5/06C12N5/10C12N5/074
CPCC07K14/4702C12N5/0696C12N2501/60C12N2510/00C12N2501/606C12N2501/602C12N2501/603C12N2501/604C12N2740/15043
Inventor YAMANAKA, SHINYATAKAHASHI, KAZUTOSHINAKAGAWA, MASATO
Owner KYOTO UNIV
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