Method for neural differentiation of embryonic stem cells using protease passaging techniques
First Claim
1. A human pluripotent embryonic stem cell culture, wherein the cells of the culture do not express SSEA1, express SSEA3, SSEA4, Oct4, Tra-1-60, Tra-1-80, and express nestin substantially uniformly.
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Accused Products
Abstract
The present invention provides methods for human pluripotent cell culturing and for neural cell production. More particularly, the present invention provides culturing methods employing dissociating cell cultures to an essentially single cell culture, such as by employing antibody selection and bulk passaging treatments utilizing the subsequent application of Collagenase and trypsin. In certain embodiments, the cells are further treated with essentially serum free MEDII conditioned medium, proline, or minimal medium, and are optionally treated with amphiphilic lipid compounds for the generation of human neural cells from pluripotent human cells. In certain embodiments, the cells cultured using these methods have an abnormal karyotype.
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Citations
74 Claims
- 1. A human pluripotent embryonic stem cell culture, wherein the cells of the culture do not express SSEA1, express SSEA3, SSEA4, Oct4, Tra-1-60, Tra-1-80, and express nestin substantially uniformly.
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10. A method of culturing a human pluripotent embryonic stem cell comprising,
a) selecting a human pluripotent cell using an anti-SSEA4 antibody; - and
b) maintaining a culture of the cell by passaging the cell using a protease treatment, wherein the cells of the culture do not express SSEA1, express SSEA3, SSEA4, Oct4, Tra-1-60, Tra-1-80, and express nestin substantially uniformly. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36)
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28. The method of claim 0, wherein the minimal medium comprises proline at a concentration from approximately 50 μ
- M to approximately 250 μ
M.
- M to approximately 250 μ
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37. A method of culturing a human pluripotent embryonic stem cell comprising,
a) providing a human pluripotent embryonic stem cell culture; -
b) passaging the cell culture using a protease treatment to thereby disperse the cell to an essentially single cell culture; and
c) culturing the essentially single cell culture in the presence of a feeder cell, a conditioned medium, or a minimal medium to thereby culture the human pluripotent embryonic stem cell. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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51. A method of producing a human pluripotent embryonic stem cell culture enriched in neural cells comprising,
a) providing a human pluripotent embryonic stem cell culture; -
b) passaging the cell culture using a protease treatment to thereby disperse the cell culture to an essentially single cell culture;
c) culturing the essentially single cell culture in the presence of a feeder cell, a conditioned medium, or a minimal medium; and
d) forming an embryoid body comprising the essentially single cell culture by culturing the cell culture with an essentially serum free medium, to thereby produce the human cell culture enriched in neural cells. - View Dependent Claims (52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74)
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Specification