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METHOD FOR DIFFERENTIATING PLURIPOTENT STEM CELLS INTO DESIRED CELL TYPE

Foreign code F160008885
File No. (S2015-0777-N0)
Posted date Oct 25, 2016
Country WIPO
International application number 2016JP057420
International publication number WO 2016143826
Date of international filing Mar 9, 2016
Date of international publication Sep 15, 2016
Priority data
  • P2015-046318 (Mar 9, 2015) JP
Title METHOD FOR DIFFERENTIATING PLURIPOTENT STEM CELLS INTO DESIRED CELL TYPE
Abstract Provided is a method for predicting the directionality of cell differentiation caused by induced expression of a transcription factor and differentiating mammalian pluripotent stem cells into a desired cell type. The inventors newly created a human gene expression correlation matrix using human cells and furthermore introduced a transcription factor cocktail selected from the matrix into human pluripotent stem cells, and thereby confirmed that differentiation into a desired cell type is possible and perfected the present invention.
Outline of related art and contending technology BACKGROUND ART
(Transcription factor network) the network of transcription factors (TF), determine the identity cells, a combination of a plurality of transcription factor by over expression, can be modified. Results in a particular cell differentiation transcription factor selects the combination, proved, transcription factor of the possible combinations over about 2,000 species and the difficulties for many. One purpose of regenerative medicine is the embryonal stem cells (ES) 1 and an induced pluripotent stem (iPS) cells from pluripotent stem cells such as the desired type of differentiated cells generated in {non-patent document 1: Nature 292, 154-156 (1981), non-patent document 2: Proc Natl Acad Sci U S A 78, 7634-7638 (1981), non-patent document 3: Science 282, 1145-1147 (1998), non-patent document 4: Cell 126, 663-676 (2006) }. However, a wide variety of enormous and the adjustment mechanism of the transcription factor is complex, large in order to find the right combination of transcription factors has been a problem.
To facilitate the transcription factor network analysis, loss of function, knock-out or mouse ES cells that suppressed transcription factor {non-patent document 5: Nat Genet 36, 543-544 (2004), non-patent document 6: Nature 442, 533-538 (2006), non-patent document 7: Cell 128, 9-13 (2007), non-patent document 8: Sci Rep 3, 1390 (2013) } system biology approach to been applied {non-patent document 9: Annu Rev Cell Dev Biol 26, 721-744 (2010) }, then, phenotypic analysis or extensive transcriptome analysis is performed. However, a combination of the transcription factor by inducing forcibly, identification information of the modified cells can be achieved in this manner it is significant, the acquisition of function, over-expression approach is more preferred that the transcription factor {non-patent document 10: Cell 51, 987-1000 (1987), non-patent document 4: Cell 126, 663-676 (2006), non-patent document 11: Proc Natl Acad Sci U S A 105, 6057-6062 (2008), non-patent document 12: Nature 468, 521-526 (2010), non-patent document 13: Nature 463, 1035-1041 (2010), non-patent document 14: Nature 476, 224-227 (2011), non-patent document 15: Cell Stem Cell 9, 205-218 (2011), non-patent document 16: Nature 475, 390-393 (2011), non-patent document 17: Nature 475, 386-389 (2011) }. Therefore, NIA Mouse ES Cell Bank {non-patent document 18: Cell Stem Cell 5, 420-433 (2009), non-patent document 19: Sci Rep 1, 167 (2011) } is established, in this case, each one of the 137 transcription factor, i.e. the entire transcription factor encoded by the mouse genome (1500-2000 type of transcription factor) of 7-10% {non-patent document 20: Biochem Biophys Res Commun 322, 787-793 (2004) } is derived by a method in a tetracycline regulated. Each transcription factor and then forcibly induced expression after 48, a wide range of these ES cell lines in each of the gene expression profile was measured (that is the transcriptome) {non-patent document 19: Sci Rep 1, 167 (2011) }. On the other hand, various cell types, tissue, organ or the whole of the expression amount of the expression profile, can be utilized as the database of public domain. One of such systems is the expression profile of cell types in various Genomics Institute of the Novartis Research Foundation(GNF) is {non-patent document 21: Genome Biol 10, R130 (2009), non-patent document 22: Proc Natl Acad Sci U S A 99, 4465-4470 (2002) }. Is obtained in the experiment described above transcription factor-induced gene expression profile and comparing the gene expression profile of GNF by, matrix showing the correlation of gene expression levels (gene expression correlation matrix) was prepared.
The differentiation state of cells (cell differentiation), the cells express a set of a particular transcription factor by the expression levels thereof are considered to be defined. Transcription factor, and the factors that control gene expression directly, such as a promoter or enhancer binds to a region of controlling transcription, the genetic information of the DNA is transferred to promote RNA processes, or suppressed, cell-specific gene that plays an important role to form a network. In recent years, an induced pluripotent stem cell (iPS cell) using any cells differentiation techniques have been developed are intensively studied, the differentiation efficiency of mixing of a low or a different lineage in question. Therefore, the determination of terminal differentiation in a genetically modified cell differentiation can be identification of the transcription factor is a very important issue.
(Mouse transcription factor network) the inventors of the present invention, to determine cell lineage differentiation of mouse transcription factor to elucidate the structure of the network, so as to be a transcription factor capable of inducing mouse mouse ES cell bank NIA expression (transcription factor 137 genetic Anodic cell lines) are to establish {non-patent document 18: Cell Stem Cell 5, 420-433 (2009), non-patent document 19: Sci Rep 1, 167 (2011) }. In these cell lines, using Tet-off system, doxycycline can be removed from the culture medium by a single transcription factor, can be quickly forced expression and strong. The inventors have found that, using these cell lines, inducing gene expression after 48 of the amounts of transcripts of the test were covered by the microarray. The resulting gene expression profiles, each mouse organ, in comparison with gene expression (gene expression correlation matrix) by, caused by the induction of expression of a single transcription factor gene expression pattern of change, which tends to define the lineage was observed clearly could be. As a result, induction of expression of the transcription factor caused by the orientation of cell differentiation can be predicted with considerable accuracy is checked.
Scope of claims (In Japanese)[請求項1]
以下の(1)~(5)のいずれか1以上の転写因子を哺乳類由来の多能性幹細胞に導入する工程を含む、多能性幹細胞を神経系細胞に分化する方法。
(1)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される5の転写因子
(2)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される4の転写因子
(3)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される3の転写因子
(4)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される2の転写因子
(5)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される1の転写因子
[請求項2]
前記神経系細胞は、運動神経を含む請求項1に記載の分化する方法。
[請求項3]
前記運動神経は、運動神経に存在する細胞である請求項1又は2に記載の分化する方法。
[請求項4]
以下の(1)~(5)のいずれか1以上の転写因子を含む哺乳類由来の多能性幹細胞を神経系細胞に分化可能な神経系細胞分化誘導剤。
(1)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される5の転写因子
(2)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される4の転写因子
(3)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される3の転写因子
(4)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される2の転写因子
(5)NEUROG1、NEUROG2、NEUROG3、NEUROD1及びNEUROD2から選択される1の転写因子
[請求項5]
前記神経系細胞は、末梢性の運動神経である請求項4に記載の分化誘導剤。
[請求項6]
前記運動神経は、運動神経に存在する細胞である請求項4又は5に記載の分化誘導剤。
[請求項7]
前記転写因子は、mRNA、合成mRNA、核酸又はタンパク質である請求項4~6のいずれか1に記載の分化誘導剤。
[請求項8]
以下の(1)~(5)のいずれか1以上の転写因子を哺乳類由来の多能性幹細胞に導入する工程を含む、多能性幹細胞を肝芽細胞及び/又は肝細胞に分化する方法。
(1)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される1の転写因子
(2)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される2の転写因子
(3)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される3の転写因子
(4)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される4の転写因子
(5)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される5の転写因子
[請求項9]
以下の(1)~(5)のいずれか1以上の転写因子を含む哺乳類由来の多能性幹細胞を肝芽細胞及び/又は肝細胞に分化可能な肝芽細胞及び/又は肝細胞分化誘導剤。
(1)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される1の転写因子
(2)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される2の転写因子
(3)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される3の転写因子
(4)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される4の転写因子
(5)TGIF、TCF4、PITX2、SALL4及びMEIS1から選択される5の転写因子
[請求項10]
以下の(1)~(7)のいずれか1以上の転写因子を哺乳類由来の多能性幹細胞に導入する工程を含む、多能性幹細胞を造血幹細胞及び/又は血液細胞に分化する方法。
(1)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される1の転写因子
(2)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される2の転写因子
(3)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される3の転写因子
(4)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される4の転写因子
(5)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される5の転写因子
(6)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される6の転写因子
(7)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される7の転写因子
[請求項11]
以下の(1)~(7)のいずれか1以上の転写因子を含む哺乳類由来の多能性幹細胞を造血幹細胞及び/又は血液細胞に分化可能な造血幹細胞及び/又は血液系細胞分化誘導剤。
(1)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される1の転写因子
(2)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される2の転写因子
(3)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される3の転写因子
(4)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される4の転写因子
(5)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される5の転写因子
(6)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される6の転写因子
(7)CDYL2、ETS2、SPI1、OVOL2、CDX2、CEBPB及びSALL4から選択される7の転写因子
[請求項12]
転写因子であるSOX9を哺乳類由来の多能性幹細胞に導入する工程を含む、多能性幹細胞を軟骨細胞に分化する方法。
[請求項13]
転写因子であるSOX9を含む哺乳類由来の多能性幹細胞を軟骨細胞に分化可能な軟骨細胞分化誘導剤。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KEIO UNIVERSITY
  • Inventor
  • KO,Minoru
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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