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Method for producing tissue cells from pluripotent stem cells derived from iris pigment epithelial cells of animal and tissue cell obtained by method

外国特許コード F110005449
整理番号 K05205US
掲載日 2011年9月6日
出願国 アメリカ合衆国
出願番号 55978304
公報番号 20060141621
公報番号 8796014
出願日 平成16年6月10日(2004.6.10)
公報発行日 平成18年6月29日(2006.6.29)
公報発行日 平成26年8月5日(2014.8.5)
国際出願番号 JP2004008120
国際公開番号 WO2004111212
国際出願日 平成16年6月10日(2004.6.10)
国際公開日 平成16年12月23日(2004.12.23)
優先権データ
  • 特願2003-166684 (2003.6.11) JP
  • 2004WO-JP08120 (2004.6.10) WO
発明の名称 (英語) Method for producing tissue cells from pluripotent stem cells derived from iris pigment epithelial cells of animal and tissue cell obtained by method
発明の概要(英語) (US8796014)
A method for producing tissue cells derived from iris pigmented epithelial cells of an animal, and tissue cells obtained by the method are provided.
The method and the tissue cells solve problems such as immunological rejection in cell transplantation, ethical issues, and unbalance between the demand and supply of transplant cell sources.
In the method of the present invention for producing the tissue cells, first, the iris pigmented epithelial cells isolated from an eyeball of an animal are selectively cultured according to a floated coagulated mass culturing technique so as to obtain pluripotent stem cells.
Thereafter, the pluripotent stem cells are cultured by using, for example, serum so as to produce various tissue cells.
特許請求の範囲(英語) [claim1]
1. A method for producing tissue cells wherein the tissue cells are myocardial cells, the method comprising the steps of: (i) an iris-tissue-extirpating step of extirpating iris tissue from the eyeball of the animal;
(ii) an iris-pigmented-epithelial-cell-separating step of separating iris pigmented epithelium from the iris tissue thus extirpated;
(iii) dissociating the separated iris pigmented epithelium using a trypsin solution;
(iv) obtaining pluripotent stem cells by selectively culturing iris pigment epithelial cells by a floated coagulated mass culturing technique, the iris pigmented epithelial cells separated by the steps (i)-(iii) being isolated from an eyeball of an animal, the floated coagulated mass culturing technique comprising culturing the isolated iris pigmented epithelial cells in a culturing media with rotation, the culturing media comprising serum free medium, an N2 supplement, and at least one factor selected from the group consisting of fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and stem cell factor (SCF); and
(v) obtaining myocardial cells from the pluripotent stem cells by differentiating the pluripotent stem cells into myocardial cells by culturing the pluripotent stem cells under differentiation-inducing conditions comprising culturing the pluripotent stem cells for one to two months in a culture medium comprising fetal calf serum, avian serum, epidermal growth factor (EGF), and fibroblast growth factor 2 (FGF2).
[claim2]
2. The method according to claim 1, wherein the animal is a chicken, a mouse, a rat, or a human.
[claim3]
3. The method according to claim 1, wherein the animal is a postnatal individual animal.
[claim4]
4. The method according to claim 1, wherein the pluripotent stem cells are Oct-3/4 positive and/ or tridermic differentiatable.
[claim5]
5. The method according to claim 1, wherein the iris-tissue-extirpating step includes: an iris-tissue-excising stage of excising only iris tissue from the eyeball of the animal;
an enzyme treatment stage of subjecting the excised iris tissue to enzyme treatment; and
an iris-tissue-restoring stage of restoring, by using a culture medium containing serum, the iris tissue weakened by the enzyme treatment.
[claim6]
6. The method according to claim 5, wherein in the enzyme treatment step, the iris tissue is treated in a dispase solution and then treated in an EDTA solution.
[claim7]
7. The method according to claim 5, wherein in the iris-restoring step, the iris tissue is treated in a culture medium comprising fetal calf serum.
[claim8]
8. The method of claim 1, further comprising testing for expression of at least one gene specific for myocardial cells.
[claim9]
9. The method of claim 8, wherein the gene specific for myocardial cells is selected from the group consisting of GATA4, Nkx2.5, cMyBP, and myosin.
[claim10]
10. The method of claim 1, further comprising examining the pluripotent stem cells, after step (iv), for absence or presence of expression of an endodermal marker gene, a mesodermal marker gene, and an ectodermal marker gene.
[claim11]
11. The method of claim 10, wherein the endodermal marker gene is fetoprotein alpha , the mesodermal marker gene is myosin or MEF2, and the ectodermal marker is pax 6 or tubulin J.
  • 発明者/出願人(英語)
  • KOSAKA MITSUKO
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 435/325
  • 435/6.11
  • 435/7.1
  • 435/377
参考情報 (研究プロジェクト等) PRESTO Time's Arrow and Biosignaling AREA
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