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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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0008]The present invention provides induced pluripotent stem (iPS) cells derived by nuclear reprogramming of a somatic cell. The present invention also provides methods for reprogramming of a differentiated cell without using eggs, embryos, or embryonic stem (ES) cells. The present invention also provides nuclear reprogramming factors for induction of pluripotent stem cells. The disclosed methods and nuclear reprogrammi

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 t

Method used

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  • Nuclear reprogramming factor and induced pluripotent stem cells
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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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