IPC분류정보
국가/구분 |
United States(US) Patent
등록
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국제특허분류(IPC7판) |
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출원번호 |
US-0211959
(2011-08-17)
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등록번호 |
US-9528090
(2016-12-27)
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발명자
/ 주소 |
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출원인 / 주소 |
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대리인 / 주소 |
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인용정보 |
피인용 횟수 :
3 인용 특허 :
74 |
초록
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The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells, wherein greater than 85% of the cells in the population express markers characteristi
The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells, wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage.
대표청구항
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1. An in vitro cell culture comprising an isolated population of cells and a culture medium suitable for differentiating pluripotent stem cells, wherein greater than 85% of the cells are definitive endoderm cells,wherein said population of cells is obtained by differentiating in vitro pluripotent st
1. An in vitro cell culture comprising an isolated population of cells and a culture medium suitable for differentiating pluripotent stem cells, wherein greater than 85% of the cells are definitive endoderm cells,wherein said population of cells is obtained by differentiating in vitro pluripotent stem cells,wherein the culture medium lacks serum and is supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1, andwherein said definitive endoderm cells express CXCR4 and do not express CD9. 2. The in vitro culture of claim 1, wherein the isolated population of cells is obtained without further purifying the cells after differentiation. 3. The in vitro culture of claim 1, wherein the medium is further supplemented with activin A and Wnt-3. 4. The in vitro culture of claim 1, wherein the medium is chemically-defined. 5. A method for generating a population of cells wherein greater than 85% of the cells in the population are definitive endoderm cells, comprising the steps of: a. culturing a population of pluripotent stem cells; andb. differentiating the population of pluripotent stem cells to a population of cells wherein greater than 85% of the cells in the population are definitive endoderm cells in medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1. 6. The method of claim 5, wherein the population of pluripotent stem cells is differentiated in the medium lacking serum and supplemented with BSA and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1 for a period of at least 6 days. 7. The method of claim 5, wherein the population of pluripotent stem cells is differentiated in the medium lacking serum and supplemented with BSA and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1 for a period of at least 7 days. 8. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and from about 1 ng/ml to about 50 ng/ml of IGF-1. 9. The method claim 5, wherein the population of cells is obtained without further purifying the cells after differentiation. 10. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and insulin. 11. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, from about 5 ng/ml to about 500 ng/ml of GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1. 12. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with from about 0.5 to about 2% BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1. 13. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and from about 1 ng/ml to about 100 ng/ml of insulin. 14. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with about 2% BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and from about 1 ng/ml to about 50 ng/ml of IGF-1. 15. The method of claim 5, wherein the medium is chemically-defined. 16. An in vitro culture comprising an isolated population of cells in which greater than 85% of the cells are definitive endoderm cells and a cell culture medium, wherein said population of cells is obtained by differentiating pluripotent stem cells into definitive endoderm cells, andwherein the cell culture medium lacks serum and is supplemented with BSA, GDF-8, 14- and Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1-8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and either insulin or from about 1 ng/ml to about 50 ng/ml of IGF-1. 17. The in vitro culture of claim 16, wherein the population is obtained by differentiating pluripotent stem cells into definitive endoderm cells by treating the pluripotent stem cells with the medium lacking serum and supplemented with BSA, GDF-8, 14- and Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and either insulin or from about 1 ng/ml to about 50 ng/ml of IGF-1.
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