Techniques for growth and differentiation of human pluripotent stem cells
First Claim
1. A composition comprising proliferating primate pluripotent stem (pPS) cells, which is essentially free of feeder cells.
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Abstract
This disclosure provides an improved system for culturing human pluripotent stem (pPS) cells in the absence of feeder cells. The role of the feeder cells can be replaced by supporting the culture on an extracellular matrix, and culturing the cells in a conditioned medium. Permanent cell lines are provided that can produce conditioned medium on a commercial scale. Methods have also been discovered to genetically alter pPS cells by introducing the cells with a viral vector or DNA/lipid complex. The system described in this disclosure allows for bulk proliferation of pPS cells for use in studying the biology of pPS cell differentiation, and the production of important products for use in human therapy.
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Citations
32 Claims
- 1. A composition comprising proliferating primate pluripotent stem (pPS) cells, which is essentially free of feeder cells.
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4. A method for culturing primate pluripotent stem (pPS) cells, comprising culturing pPS cells in a growth environment essentially free of feeder cells but containing conditioned medium produced by collecting medium from a culture of feeder cells.
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5. A method for producing a conditioned medium suitable for culturing primate pluripotent stem (pPS) cells in a growth environment essentially free of feeder cells, comprising:
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a) conditioning medium by culturing cells in the medium, wherein the cells are a euploid cell line that can proliferate in culture for at least 60 days; and
b) harvesting the conditioned medium. - View Dependent Claims (6)
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8. A human cell line obtained by differentiating a culture of human embryonic stem (hES) cells into a population of differentiated cells that comprises fibroblast-like cells, and then selecting fibroblast-like cells from the culture;
- wherein conditioned medium produced by harvesting medium from a culture of the fibroblast-like cells supports growth of pPS cells in a culture environment essentially free of feeder cells.
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11. A method for producing differentiated cells from a donor culture of undifferentiated primate pluripotent stem (pPS) cells, comprising:
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a) preparing a suspension of cells from the undifferentiated donor culture;
b) replating and culturing the suspended cells on a solid surface so that they differentiate without forming embryoid bodies; and
c) harvesting differentiated cells from the solid surface. - View Dependent Claims (13, 14, 16)
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12. A method for producing differentiated cells from a donor culture of primate pluripotent stem (pPS) cells, comprising:
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a) providing a culture of primate pluripotent stem (pPS) cells that is essentially free of feeder cells;
b) changing the medium in which the cells are cultured; and
c) harvesting differentiated cells after culturing for a time in the changed medium.
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17. A method for producing a polynucleotide comprising a nucleotide sequence contained in an mRNA that is expressed at a different level in committed or differentiated cells compared with undifferentiated primate pluripotent stem (pPS) cells, the method comprising:
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a) determining the level of expression of a plurality of mRNAs in committed or differentiated cells, in comparison to the level of expression of the same mRNAs in undifferentiated pPS cells;
b) identifying an mRNA expressed at a different level in the committed or differentiated cells, relative to the undifferentiated pPS cells; and
c) preparing a polynucleotide comprising a nucleotide sequence of at least 30 consecutive nucleotides contained in the identified mRNA.
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19. A method of producing genetically altered primate pluripotent stem (pPS) cells, comprising:
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a) providing a composition of pPS cells on a layer of feeder cells that are drug-resistant;
b) transferring a polynucleotide into pPS cells in the composition; and
c) selecting genetically altered cells in the composition using the drug to which the feeder cells are resistant.
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- 21. A population of primate pluripotent stem (pPS) cells, in which at least 25% of the undifferentiated pPS cells have been stably transfected with a polynucleotide, or are the progeny of such cells that have inherited the polynucleotide.
- 23. A method of producing an mRNA preparation or a cDNA library from primate pluripotent stem (pPS) cells before or after differentiation, comprising providing a culture of undifferentiated pPS cells essentially free of feeder cells, optionally permitting the pPS cells to differentiate, and isolating mRNA from the undifferentiated or differentiated cells.
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27. A cDNA library of at least 1,000 genes expressed at the mRNA level in either undifferentiated pPS cells, or cells differentiated from pPS cells, wherein the library is essentially free of cDNA of other vertebrates.
Specification