WO2011071118A1 - ニトロビンを含む多能性幹細胞の心筋細胞への分化促進剤 - Google Patents
ニトロビンを含む多能性幹細胞の心筋細胞への分化促進剤 Download PDFInfo
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
Definitions
- the present invention relates to an agent for promoting differentiation of pluripotent stem cells into cardiomyocytes, a method for inducing differentiation of pluripotent stem cells into cardiomyocytes, and a method for producing cardiomyocytes from pluripotent stem cells.
- the method of inducing differentiation of pluripotent stem cells into desired cells is an essential technique for realizing regenerative medicine. Further, differentiation-induced cells obtained by such a method are expected to be useful not only for regenerative medicine but also for in vitro drug efficacy evaluation tests and drug safety tests. Since heart disease is currently the second leading cause of death in Japanese people, it is particularly important to establish a method for inducing differentiation into cardiomyocytes that can be used for regenerative medicine to treat heart disease and for evaluating the efficacy of heart disease. Expectation is put. In addition, since there are many drugs that cause serious side effects such as heart failure and arrhythmia on the heart, there is a need for a uniform supply of cardiomyocytes that can be used in cardiotoxicity tests.
- Non-patent Document 1 a method of co-culturing END2 cells, which are mouse-derived support cells, and human ES cells has been reported (Non-patent Document 1).
- END2 cells which are mouse-derived support cells
- human ES cells has been reported (Non-patent Document 1).
- its differentiation efficiency is not sufficient, and since mouse-derived END2 cells are easily mixed into human cardiomyocytes, there is a problem that it is difficult to obtain pure human cardiomyocytes.
- cytokines fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), Dickkopf-
- bFGF fibroblast growth factor
- BMP4 bone morphogenetic protein 4
- VEGF vascular endothelial growth factor
- DKK1 vascular endothelial growth factor
- DKK1 activin A
- the present inventors examined the differentiation promoting effect from monkey ES cells to cardiomyocytes for 9600 kinds of library compounds. As a result, it was found that nitrobin, a low molecular weight compound known as a livestock antibiotic / growth promoter, exhibits a very high myocardial differentiation efficiency compared to known myocardial differentiation promoting factors, leading to the completion of the present invention. It was.
- the present invention provides an agent for promoting differentiation of pluripotent stem cells into cardiomyocytes containing nitrobin.
- the present invention also provides a method for inducing differentiation of pluripotent stem cells into cardiomyocytes, comprising culturing the pluripotent stem cells in a medium containing nitrobin.
- the present invention also provides a method for producing cardiomyocytes from pluripotent stem cells, comprising culturing pluripotent stem cells in a medium containing nitrobin.
- pluripotent stem cells can be induced to differentiate into cardiomyocytes without using feeder cells, and pure cardiomyocytes can be obtained.
- the present invention makes it possible to produce cardiomyocytes by inducing differentiation into cardiomyocytes with high efficiency and low cost.
- the present invention relates to mass production of homogeneous and mature human cardiomyocytes for use in high-throughput screening for QT prolongation tests important as drug safety tests, or for evaluation of drug efficacy against heart diseases, and transplanted cardiomyocytes for heart diseases etc. Is particularly useful in the production of
- a screening screening method for a myocardial differentiation promoting substance Screening system and nitrobin detection.
- GFP expression upon administration of nitrobin final concentrations 1, 5, 20 ⁇ M
- the horizontal axis indicates the concentration of nitrobin.
- the vertical axis of the left graph shows the amount of GFP fluorescence at the time of nitrobin administration when dimethyl sulfoxide (DMSO) administration (control) is 100%.
- the vertical axis of the right graph shows the number of beating colonies per well.
- GFP expression upon administration of nitrobin final concentration 5 ⁇ M in human ES cells.
- nitrobin final concentration 5 ⁇ M
- nitrobin final concentration 5 ⁇ M
- cytokines bFGF, BMP4, VEGF, DKK1, activin A
- expression of GFP upon administration of cytokine desame as above
- nitrobin final concentration 5 ⁇ M
- the vertical axis of the left graph shows the amount of GFP fluorescence when nitrobin and / or cytokine is administered when DMSO administration (control) is 100%.
- the vertical axis of the right graph shows the number of beating colonies per well.
- the vertical axis shows the expression level when nitrobin and / or cytokine is administered when DMSO administration (control) is 100%.
- the present invention relates to an agent for promoting differentiation of pluripotent stem cells into cardiomyocytes, comprising nitrobin.
- Nitrobin has the chemical formula It is a low molecular compound having (molecular formula: C14H12N6O6). Nitrobin is a known compound and can be produced by conventional organic synthesis methods. It can also be obtained from Kanto Chemical Co., Inc. (Tokyo, Japan).
- pluripotency means pluripotency capable of differentiating into all cells constituting an adult and self-replication capable of maintaining the pluripotency even after cell division. It means a cell having an ability.
- the “pluripotent stem cell” includes embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells).
- the species of “pluripotent stem cells” is not particularly limited, but is preferably a mammal, more preferably a rodent or a primate.
- the present invention is particularly suitable for monkey or human pluripotent stem cells.
- ES cells are pluripotent stem cells derived from early embryos, and can be established from the inner cell mass of blastocysts or epiblasts of early embryos after implantation.
- ES cells include humans (Thomson J. A. et al., Science 282: 1145-1147 (1998); primates such as rhesus monkeys and marmosets) (Thomson J. A. et al., Proc. Natl. Acad. Sci. USA 92: 7844-7848 (1995); Thomson J. A. et al., Biol. Reprod. 55: 254-259 (1996)); rabbit (special table 2000-508919); hamster (Doetshman T. et al., Dev. Biol.
- EG cells are pluripotent stem cells derived from primordial germ cells, and examples include human EG cells (Shamblott, et al., Proc. Natl. Acad. Sci USA 92: 7844-7848 (1995)).
- iPS cells mean pluripotent stem cells derived from cells other than pluripotent stem cells such as somatic cells and tissue stem cells.
- pluripotent stem cells such as somatic cells and tissue stem cells.
- iPS cells are included in the “iPS cells” of the present invention as long as they are artificially derived from cells other than pluripotent stem cells.
- iPS cells have genetic information derived from somatic cells, for example. Therefore, cardiomyocytes differentiated from patient-derived iPS cells with the differentiation inducer of the present invention are useful not only for regenerative medicine, but also for studying the effectiveness and side effects of drugs for individual patients.
- cardiomyocytes differentiated from iPS cells derived from heart disease patients are expected to have genetic information unique to patients with heart diseases, and are also used for screening drugs for heart diseases caused by gene abnormalities. Preferably used.
- the differentiation promoting agent of the present invention is added to the myocardial differentiation medium of pluripotent stem cells so that the final concentration of nitrobin is 1 to 5 ⁇ M, for example.
- the myocardial differentiation medium is not particularly limited as long as it is a composition generally used for myocardial differentiation of pluripotent stem cells.
- myocardial differentiation medium based on IMDM medium used in the examples of the present application.
- DMEM-based myocardial differentiation medium (DMEM / F12 medium (Sigma) 200 ml, fetal bovine serum (GIBCO) 50 ml, MEM non-essential amino acid solution (Sigma) 2.5 ml, penicillin-streptomycin (GIBCO) 2.5 ml, 200 mM L-glutamine 2.5 ml, 2-mercaptoethanol 2 ⁇ l, or StemPro-34SFM (GIBCO) + BMP4 (10 ng / ml) medium is exemplified. In this case, it is not necessary to use feeder cells such as END2 cells, etc.
- the timing of addition of the differentiation promoting agent of the present invention depends on the type of pluripotent stem cell used.
- the culture is started 6 to 6 after the start of the culture in the myocardial differentiation medium. It may be added on day 14.
- the agent for promoting differentiation of the present invention may be used together with other substances having a myocardial differentiation promoting action, for example, cytokines such as bFGF, BMP4, VEGF, DKK1, and activin A.
- cytokines such as bFGF, BMP4, VEGF, DKK1, and activin A.
- the addition amount of other substances and the timing of addition may be appropriately determined.
- the cardiomyocyte differentiation induction method and cardiomyocyte production method of the present invention are characterized by culturing pluripotent stem cells in a medium containing nitrobin.
- the method of the present invention comprises culturing pluripotent stem cells in a myocardial differentiation medium, and adding nitrobin to a final concentration of 1 to 5 ⁇ M 6 to 14 days after the start of culture in the myocardial differentiation medium. And by confirming differentiation of pluripotent stem cells into cardiomyocytes on day 18 of culture.
- what is necessary is just to determine suitably the optimal time according to the cell to be used for the time of nitrobin addition and the differentiation confirmation to a cardiac muscle cell.
- cytokines such as bFGF, BMP4, VEGF, DKK1, and activin A
- nitrobin cytokines
- action a substance which has another myocardial differentiation promotion effect
- action a substance which has another myocardial differentiation promotion effect
- action a substance which has another myocardial differentiation promotion effect
- action a substance which has another myocardial differentiation promotion effect
- action a substance which has another myocardial differentiation promotion effect
- the culture may be performed under normal animal cell culture conditions, for example, but not limited to, in an atmosphere of a temperature of 37 ° C., a humidity of 100%, and a carbon dioxide concentration of 5%.
- Differentiation into cardiomyocytes can be confirmed by, for example, the number of beating cardiomyocytes and the expression level of the ⁇ -MHC gene, which is a myocardial differentiation marker.
- the cardiomyocytes obtained by the method of the present invention can be used for in vitro drug safety tests or as transplanted cardiomyocytes for heart diseases and the like.
- FIG. 1 Screening for Substances that Promote Myocardial Differentiation of Monkey ES Cells
- GFP green fluorescent protein
- nitrobin On the sixth day of culture, nitrobin was added so that the final concentrations were 1 ⁇ M, 5 ⁇ M, and 20 ⁇ M. On the 18th day of culture, the amount of GFP fluorescence and the number of heartbeat beating colonies were measured. By administration of nitrobin, the GFP fluorescence amount increased by about 5 times and the number of myocardial beating colonies increased by about 10 times (FIGS. 3-1, 3-2). A final concentration of 5 ⁇ M was most effective.
- GFP green fluorescent protein
- Nitrobin promotes myocardial differentiation in human ES cells
- a vector expressing green fluorescent protein (GFP) was introduced into a human ES cell line (Kh-1 strain) in the same manner as Example 1 were used.
- Human ES cells were seeded and cultured in 6-well plates (Asahi Glass / 5816-006: Ezview culture plate) at 1.2 ⁇ 10 6 cells / well. The same medium as in Example 1 was used.
- nitrobin was added so that the final concentrations were 1 ⁇ M, 5 ⁇ M, and 20 ⁇ M.
- the amount of GFP fluorescence was measured. As a result, an increase in the amount of GFP fluorescence was also observed in human ES cells by administration of nitrobin (FIGS. 4-1, 4-2).
- cytokines bFGF, BMP4, VEGF, DKK1, Activin A
- bFGF, BMP4, VEGF, DKK1, Activin A cytokines known as factors that promote myocardial differentiation and nitrobin
- GFP fluorescence increase GFP fluorescence and increase myocardial beating colony count
- the increase in the expression level of troponin T was compared.
- cells in which a vector expressing green fluorescent protein (GFP) was introduced into monkey ES cells (cynomolgus monkey CMK6.4 strain) were used.
- the same medium as in Example 1 was used.
- Monkey ES cells were seeded in a 6-well plate, nitrobin (final concentration 5 ⁇ M) was cultured on days 6 to 14, and cytokine mixture (bFGF, BMP4, VEGF, DKK1, activin A) (final concentration) was cultured on days 1 to 14 5 ng / ml, 10 ng / ml, 10 ng / ml, 150 ng / ml, 3 ng / ml) were administered, and the amount of GFP fluorescence, the number of heartbeat beating colonies, and the expression level of troponin T were measured on the 18th day of culture.
- bFGF, BMP4, VEGF, DKK1, activin A final concentration
- the nitrobin alone administration group showed a higher effect than the cytokine mixture administration group (FIGS. 5-1 to 5-3). Further, when nitrobin and cytokine were used in combination, a synergistic effect was observed in the increase in the amount of GFP fluorescence and the increase in the expression level of troponin T (FIGS. 5-1, 5-3).
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Abstract
Description
iPS細胞は、例えば体細胞から作成される場合にはその体細胞由来の遺伝情報を有している。従って、本発明の分化誘導剤により患者由来のiPS細胞から分化誘導された心筋細胞は、再生医療のみならず個別の患者に対する薬剤の有効性や副作用の検討等に有用である。また、心臓疾患患者由来のiPS細胞から分化誘導された心筋細胞は心臓疾患を有する患者特有の遺伝情報を有していることが期待され、遺伝子異常に起因する心臓疾患に対する薬剤のスクリーニング等にも好適に用いられる。
本発明の方法においては、他の心筋分化促進作用を有する物質、例えばbFGF、BMP4、VEGF、DKK1およびアクチビンAなどのサイトカインをニトロビンと共に用いてもよい。他の心筋分化促進作用を有する物質の添加量、添加時期は適宜定めればよい。
図1に示すように、サルES細胞の心筋分化を促進する物質の探索スクリーニングを実施した。サルES細胞株(カニクイザルCMK6.4株)に、心筋分化マーカーであるα-MHC遺伝子のプロモーターの制御下で緑色蛍光タンパク質(GFP)を発現するベクターを導入し、96ウェルプレート(Greiner/655090:96穴FIAブラックプレート)上に5.0×103細胞/ウェルにて播種し、IMDM培地を基本とした心筋分化培地(IMDM培地(Sigma l3390)200ml、ウシ胎児血清(GIBCO 10099-141)50ml、MEM non-essential amino acid solution (Sigma M7145)2.5ml、ペニシリン-ストレプトマイシン(GIBCO 15140)2.5ml、200mM L-グルタミン 2.5ml、2-メルカプトエタノール(Sigma M7522) 2μl、5N NaOH 255μlを混合したもの)で14日間培養した。培養後6~14日に、9600種類のライブラリー化合物を、1ウェルあたり1化合物(約1~5μM)投与した。培養後14日に、HCS(high contents screening)システム(モレキュラーデバイス/MetaMorph イメージングシステム)を用いてGFP発現量を測定したところ、ニトロビンを投与したウェルのGFP蛍光値が高い値を示した(図2)。
実施例1と同じくサルES細胞(カニクイザルCMK6.4株)へ緑色蛍光タンパク質(GFP)を発現するベクターを導入した細胞を用いた。サルES細胞を6ウェルプレート(旭硝子/ 5816-006 :Ezview カルチャープレート)に4.0×105細胞/ウェルにて播種して培養した。培地は実施例1と同じものを用いた。培養6日目に、最終濃度が1μM、5μM、20μMとなるようにニトロビンを添加した。培養18日目に、GFP蛍光量と心筋拍動コロニー数とを測定した。ニトロビンの投与により、最大でGFP蛍光量は約5倍、心筋拍動コロニー数は約10倍増加した(図3-1、3-2)。最終濃度5μMが最も有効であった。
実施例1と同様にしてヒトES細胞株(Kh-1株)へ緑色蛍光タンパク質(GFP)を発現するベクターを導入した細胞を用いた。ヒトES細胞を6ウェルプレート(旭硝子/ 5816-006 :Ezview カルチャープレート)に1.2×106細胞/ウェルにて播種して培養した。培地は実施例1と同じものを用いた。培養6日目に、最終濃度が1μM、5μM、20μMとなるようにニトロビンを添加した。培養18日目に、GFP蛍光量を測定した。その結果、ヒトES細胞においても、ニトロビンの投与によりGFP蛍光量の増加が観察された(図4-1、4-2)。
心筋分化を促進する因子として知られているサイトカイン(bFGF、BMP4、VEGF、DKK1、アクチビンA)とニトロビンとを用いて、GFP蛍光量の増加、心筋拍動コロニー数の増加、心筋マーカー分子であるトロポニンTの発現量増加を比較した。
実施例1と同じくサルES細胞(カニクイザルCMK6.4株)へ緑色蛍光タンパク質(GFP)を発現するベクターを導入した細胞を用いた。培地は実施例1と同じものを用いた。6ウェルプレートにサルES細胞を播種し、培養6~14日目にニトロビン(最終濃度5μM)を、培養1~14日目にサイトカイン混合物(bFGF、BMP4、VEGF、DKK1、アクチビンA)(最終濃度各5ng/ml、10ng/ml、10ng/ml、150ng/ml、3ng/ml)を投与し、培養18日目にGFP蛍光量、心筋拍動コロニー数、トロポニンT発現量を測定した。GFP蛍光量、心筋拍動コロニー数、トロポニンT発現量のいずれについても、ニトロビン単独投与群はサイトカイン混合物投与群よりも高い効果を示した(図5-1~5-3)。また、ニトロビンとサイトカインを併用することで、GFP蛍光量の増加とトロポニンT発現量の増加に相乗効果が見られた(図5-1、5-3)。
Claims (7)
- ニトロビンを含む、多能性幹細胞の心筋細胞への分化促進剤。
- 多能性幹細胞が哺乳類の細胞である、請求項1の分化促進剤。
- 多能性幹細胞が霊長類の細胞である、請求項2の分化促進剤。
- 多能性幹細胞がヒト由来の細胞である、請求項3の分化促進剤。
- ニトロビンを含む培地中で多能性幹細胞を培養することを含む、多能性幹細胞を心筋細胞に分化誘導する方法。
- ニトロビンを含む培地中で多能性幹細胞を培養することを含む、多能性幹細胞から心筋細胞を製造する方法。
- 請求項6の方法で製造された心筋細胞。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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US13/514,965 US20120244619A1 (en) | 2009-12-09 | 2010-12-09 | Composition for promoting differentiation of pluripotent stem cells into cardiac muscle cells which comprises nitrovin |
JP2011545245A JPWO2011071118A1 (ja) | 2009-12-09 | 2010-12-09 | ニトロビンを含む多能性幹細胞の心筋細胞への分化促進剤 |
EP10836038.9A EP2511366A4 (en) | 2009-12-09 | 2010-12-09 | NITROVINE MEDICINE FOR PROMOTING THE DIFFERENTIATION OF PLURIPOTENTAL STEM CELLS IN HEART MUSCLES |
CN2010800553090A CN102648274B (zh) | 2009-12-09 | 2010-12-09 | 包含双呋脒腙的促进多能干细胞分化成心肌细胞的组合物 |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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US8658425B2 (en) | 2010-08-26 | 2014-02-25 | Kyoto University | Method for promoting differentiation of pluripotent stem cells into cardiac muscle cells |
WO2014136519A1 (ja) | 2013-03-08 | 2014-09-12 | 国立大学法人京都大学 | Egf受容体阻害剤を含む多能性幹細胞の心筋分化促進剤 |
US9499790B2 (en) | 2010-08-26 | 2016-11-22 | Kyoto University | Method for promoting differentiation of pluripotent stem cells into cardiac muscle cells |
JPWO2014157257A1 (ja) * | 2013-03-25 | 2017-02-16 | 公益財団法人先端医療振興財団 | 細胞の選別方法 |
US9587220B2 (en) | 2012-01-27 | 2017-03-07 | Kyoto University | Method for inducing cardiac differentiation of pluripotent stem cell |
US10233426B2 (en) | 2014-05-30 | 2019-03-19 | Kyoto University | Method for inducing cardiac differentiation of pluripotent stem cell with low-molecular compounds |
Families Citing this family (1)
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DK2840132T3 (en) | 2013-08-22 | 2017-02-20 | Georg-August-Universität Göttingen Stiftung Öffenlichen Rechts Universitätsmedizin | Method of Manufacturing Constructed Heart Muscle (EHM) |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000508919A (ja) | 1996-04-29 | 2000-07-18 | リューベン・リサーチ・アンド・デベロップメント・ベー・ゼット・ウェー | 多能性ウサギ胚幹(es)細胞系およびそのキメラウサギの発生における使用 |
JP2003292454A (ja) * | 1990-08-23 | 2003-10-15 | Chiron Corp | 組み換えコロニー刺激因子−1の使用 |
WO2005033298A1 (ja) * | 2003-10-03 | 2005-04-14 | Keiichi Fukuda | 幹細胞から心筋細胞を分化誘導する方法 |
WO2007069666A1 (ja) | 2005-12-13 | 2007-06-21 | Kyoto University | 核初期化因子 |
WO2007126077A1 (ja) * | 2006-04-28 | 2007-11-08 | Asubio Pharma Co., Ltd. | 多能性幹細胞から心筋細胞を分化誘導する方法 |
WO2008118820A2 (en) | 2007-03-23 | 2008-10-02 | Wisconsin Alumni Research Foundation | Somatic cell reprogramming |
WO2009006997A1 (en) | 2007-06-15 | 2009-01-15 | Izumi Bio, Inc. | Human pluripotent stem cells and their medical use |
WO2009017254A1 (ja) * | 2007-07-31 | 2009-02-05 | Asubio Pharma Co., Ltd. | 心筋細胞の細胞塊作製方法及び当該心筋細胞塊の用途 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070204351A1 (en) * | 2004-01-14 | 2007-08-30 | Davidson Bruce P | Direct differentiation of cardiomyocytes from human embryonic stem cells |
-
2010
- 2010-12-09 WO PCT/JP2010/072148 patent/WO2011071118A1/ja active Application Filing
- 2010-12-09 US US13/514,965 patent/US20120244619A1/en not_active Abandoned
- 2010-12-09 JP JP2011545245A patent/JPWO2011071118A1/ja active Pending
- 2010-12-09 EP EP10836038.9A patent/EP2511366A4/en not_active Withdrawn
- 2010-12-09 CN CN2010800553090A patent/CN102648274B/zh not_active Expired - Fee Related
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2003292454A (ja) * | 1990-08-23 | 2003-10-15 | Chiron Corp | 組み換えコロニー刺激因子−1の使用 |
JP2000508919A (ja) | 1996-04-29 | 2000-07-18 | リューベン・リサーチ・アンド・デベロップメント・ベー・ゼット・ウェー | 多能性ウサギ胚幹(es)細胞系およびそのキメラウサギの発生における使用 |
WO2005033298A1 (ja) * | 2003-10-03 | 2005-04-14 | Keiichi Fukuda | 幹細胞から心筋細胞を分化誘導する方法 |
WO2007069666A1 (ja) | 2005-12-13 | 2007-06-21 | Kyoto University | 核初期化因子 |
WO2007126077A1 (ja) * | 2006-04-28 | 2007-11-08 | Asubio Pharma Co., Ltd. | 多能性幹細胞から心筋細胞を分化誘導する方法 |
WO2008118820A2 (en) | 2007-03-23 | 2008-10-02 | Wisconsin Alumni Research Foundation | Somatic cell reprogramming |
WO2009006997A1 (en) | 2007-06-15 | 2009-01-15 | Izumi Bio, Inc. | Human pluripotent stem cells and their medical use |
WO2009006930A1 (en) | 2007-06-15 | 2009-01-15 | Izumi Bio, Inc. | Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue |
WO2009007852A2 (en) | 2007-06-15 | 2009-01-15 | Izumi Bio, Inc | Multipotent/pluripotent cells and methods |
WO2009017254A1 (ja) * | 2007-07-31 | 2009-02-05 | Asubio Pharma Co., Ltd. | 心筋細胞の細胞塊作製方法及び当該心筋細胞塊の用途 |
Non-Patent Citations (25)
Title |
---|
CELL STEM CELL, vol. 3, 2008, pages 475 - 479 |
CELL STEM CELL, vol. 3, no. 5, 2008, pages 568 - 574 |
CELL STEM CELL, vol. 4, no. 5, 2009, pages 381 - 384 |
CELL, vol. 133, no. 2, 2008, pages 250 - 264 |
CELL, vol. 136, no. 3, 2009, pages 411 - 419 |
DOETSHMAN T. ET AL., DEV. BIOL., vol. 127, 1988, pages 224 - 227 |
EVANS M. J. ET AL., THERIOGENOLOGY, vol. 33, 1990, pages 125 - 128 |
EVANS M. J. ET AL., THERIOGENOLOGY, vol. 33, 1990, pages 125128 |
LESCHIK, J. ET AL.: "Cardiac commitment of primate embryonic stem cells", NAT PROTOC., vol. 3, no. 9, 2008, pages 1381 - 7, XP001538578, DOI: doi:10.1038/nprot.2008.116 |
MUMMERY, C. ET AL.: "Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm- like cells", CIRCULATION, vol. 107, no. 21, 2003, pages 2733 - 40, XP008029536, DOI: doi:10.1161/01.CIR.0000068356.38592.68 |
NATURE BIOTECHNOLOGY, vol. 26, 2008, pages 1269 - 1275 |
NATURE CELL BIOLOGY, vol. 11, 2009, pages 197 - 203 |
NATURE, vol. 454, 2008, pages 646 - 650 |
NOTARIANNI E. ET AL., J. REPROD. FERT. SUPPL., vol. 43, 1991, pages 255 - 260 |
NOTARIANNI E. ET AL., J. REPROD. FERT., vol. 40, 1990, pages 51 - 56 |
PIEDRAHITA J.A. ET AL., THERIOGENOLOGY, vol. 34, 1990, pages 879 - 891 |
SAITO S. ET AL., ROUX. ARCH. DEV. BIOL., vol. 201, 1992, pages 134 - 141 |
See also references of EP2511366A4 * |
SHAMBLOTT ET AL., PROC. NATL. ACAD. SCI USA, vol. 92, 1995, pages 7844 - 7848 |
SUKOYAN M. A. ET AL., MOL. REOROD. DEV., vol. 33, 1993, pages 418 - 431 |
TALBOT N. C. ET AL., CELL. DEV. BIOL. 29A, 1993, pages 546 - 554 |
THOMSON J. A. ET AL., BIOL. REPROD., vol. 55, 1996, pages 254 - 259 |
THOMSON J. A. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 7844 - 7848 |
THOMSON J. A. ET AL., SCIENCE, vol. 282, 1998, pages 1145 - 1147 |
YANG, L. ET AL.: "Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population", NATURE, vol. 453, no. 7194, 2008, pages 524 - 8, XP002561095, DOI: doi:10.1038/nature06894 |
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US8658425B2 (en) | 2010-08-26 | 2014-02-25 | Kyoto University | Method for promoting differentiation of pluripotent stem cells into cardiac muscle cells |
US9499790B2 (en) | 2010-08-26 | 2016-11-22 | Kyoto University | Method for promoting differentiation of pluripotent stem cells into cardiac muscle cells |
US9587220B2 (en) | 2012-01-27 | 2017-03-07 | Kyoto University | Method for inducing cardiac differentiation of pluripotent stem cell |
WO2014136519A1 (ja) | 2013-03-08 | 2014-09-12 | 国立大学法人京都大学 | Egf受容体阻害剤を含む多能性幹細胞の心筋分化促進剤 |
US10196609B2 (en) | 2013-03-08 | 2019-02-05 | Kyoto University | Composition for promoting cardiac differentiation of pluripotent stem cell comprising EGFR inhibitor |
JPWO2014157257A1 (ja) * | 2013-03-25 | 2017-02-16 | 公益財団法人先端医療振興財団 | 細胞の選別方法 |
US10233426B2 (en) | 2014-05-30 | 2019-03-19 | Kyoto University | Method for inducing cardiac differentiation of pluripotent stem cell with low-molecular compounds |
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EP2511366A4 (en) | 2014-04-30 |
US20120244619A1 (en) | 2012-09-27 |
CN102648274B (zh) | 2013-07-31 |
JPWO2011071118A1 (ja) | 2013-04-22 |
EP2511366A1 (en) | 2012-10-17 |
CN102648274A (zh) | 2012-08-22 |
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