Method for the establishment of a pluripotent human blastocyst - derived stem cell line
First Claim
1. A method for obtaining a pluripotent human blastocyst-derived stem cell line comprising:
- i) using a fertilized oocyte of grade 1 or 2 to obtain a blastocyst of grade A or B;
ii) co-culturing the blastocyst with feeder cells to establish one or more colonies of inner cell mass cells;
iii) isolating the inner cell mass cells by mechanical dissection; and
iv) co-culturing the inner cell mass cells with feeder cells to obtain a blastocyst-derived stem cell line.
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Abstract
The present invention concerns a method for the establishment of pluripotent human blastocyst-derived stem (BS) cell lines, stem cells obtained by the method, differentiation of these cells into differentiated cells, the differentiated cells, and the use of these differentiated cells in the preparation of medicaments. The undifferentiated pluripotent stem cells can be made to differentiate to a number of specialized cell types which can be utilized in the manufacture of medicaments for treating a number of conditions or pathologies involving degeneration of tissue, e.g., of the pancreas leading to, e.g., development of diabetes, or of the CNS (e.g., Alzheimer'"'"'s, Parkinson'"'"'s disease, etc.) or degeneration of the CNS caused by e.g., stroke or physical trauma.
30 Citations
61 Claims
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1. A method for obtaining a pluripotent human blastocyst-derived stem cell line comprising:
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i) using a fertilized oocyte of grade 1 or 2 to obtain a blastocyst of grade A or B;
ii) co-culturing the blastocyst with feeder cells to establish one or more colonies of inner cell mass cells;
iii) isolating the inner cell mass cells by mechanical dissection; and
iv) co-culturing the inner cell mass cells with feeder cells to obtain a blastocyst-derived stem cell line. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 57, 60, 61)
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2. A method for obtaining a pluripotent human blastocyst-derived stem cell line comprising:
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i) using a fertilized oocyte of grade 1 or 2 to obtain a blastocyst;
ii) co-culturing the blastocyst with feeder cells to establish one or more colonies of inner cell mass cells;
iii) isolating the inner cell mass cells by mechanical dissection;
iv) co-culturing the inner cell mass cells with feeder cells to obtain a blastocyst-derived stem cell line. - View Dependent Claims (58)
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3. A method for obtaining a pluripotent human blastocyst-derived stem cell line comprising:
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i) using a fertilized oocyte to obtain a blastocyst of grade A or B;
ii) co-culturing the blastocyst with feeder cells to establish one or more colonies of inner cell mass cells;
iii) isolating the inner cell mass cells by mechanical dissection; and
iv) co-culturing the inner cell mass cells with feeder cells to obtain a blastocyst-derived stem cell line. - View Dependent Claims (59)
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4. A method for obtaining a pluripotent human blastocyst-derived stem cell line comprising:
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i) using a fertilized oocyte optionally of, grade 1 or 2 to obtain a blastocyst of optionally grade A or B;
ii) co-culturing the blastocyst with feeder cells to establish for establishing one or more colonies of inner cell mass cells, iii) isolating the inner cell mass cells by mechanical dissection, iv) co-culturing the inner cell mass cells with feeder cells to obtain a blastocyst-derived stem cell line; and
v) propagation of the blastocyst-derived stem cell line culturing the stem cells with feeder cells of a density of less than about 60,000 cells per cm2.
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36. A method for producing an essentially pure preparation of insulin-producing differentiated stem cells, comprising:
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i) expanding human blastocyst-derived stem cells by growing the blastocyst-derived stem cells on an inactivated feeder cell layer in a suitable medium;
ii) generating blastocyst-derived stem cell bodies by dissociating colonies formed in step i) into smaller aggregates or individual cells, followed by transferring said aggregates or individual cells in to non-adherent containers wherein said aggregate or individual cells are incubated in a suitable medium;
iii) plating the blastocyst-derived stem cell bodies in containers in a suitable medium;
iv) selecting nestin-positive neural precursors in ITFSn medium;
v) expanding pancreatic endocrine progenitor cells in N2-medium comprising B27 media complement and basic fibroblast growth factor; and
vi) changing the medium to a basic fibroblast growth factor-free N2 medium. - View Dependent Claims (37, 38, 39, 40, 41, 55, 56)
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- 42. An essentially pure preparation of differentiated stem cells, wherein said stem cells display an expression of pancreatic cell type markers wherein said marker is at least one or more of insulin, Glut-2, Pdx-1, glucokinase, glucagons, or somatostatin.
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47. An essentially pure preparation of differentiated stem cells, wherein the stem cells express at least one of the neuronal cell type markers selected from the group consisting of:
- neuron-specific β
-III tubulin (TUJ1), NeuN, DoubleCortin, tyrosine hydroxylase, or Map 2. - View Dependent Claims (48, 53, 54)
- neuron-specific β
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49. An essentially pure preparation of stem cells obtained by:
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i) expanding human blastocyst-derived stem cells by growing the blastocyst-derived stem cells on an inactivated feeder cell layer in a suitable medium;
ii) generating blastocyst-derived stem cell bodies by dissociating colonies formed in step i) into smaller aggregates or individual cells, followed by transferring said aggregates or individual cells in to non-adherent containers wherein said aggregate or individual cells are incubated in a suitable medium; and
iii) plating the blastocyst-derived stem cell bodies in containers in a suitable medium;
iv) selecting nestin-positive neural precursors in ITFSn medium, v) expanding pancreatic endocrine progenitor cells in N2-medium comprising B27 media complement and basic fibroblast growth factor; and
vi) changing the medium to a basic fibroblast growth factor-free N2 medium.
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Specification