Direct differentiation of human pluripotent stem cells and characterization of differentiated cells
First Claim
1. A method for producing differentiated cells from a donor culture of undifferentiated primate pluripotent stem (pPS) cells, comprising:
- a) preparing a suspension of pPS 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.
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Abstract
This invention provides a system for efficiently producing differentiated cells from pluripotent cells, such as human embryonic stem cells. Rather than permitting the cells to form embryoid bodies according to established techniques, differentiation is effected directly in monolayer culture on a suitable solid surface. The cells are either plated directly onto a differentiation-promoting surface, or grown initially on the solid surface in the absence of feeder cells and then exchanged into a medium that assists in the differentiation process. The solid surface and the culture medium can be chosen to direct differentiation down a particular pathway, generating a cell population that is remarkably uniform. The methodology is well adapted to bulk production of committed precursor and terminally differentiated cells for use in drug screening or regenerative medicine.
39 Citations
22 Claims
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1. 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 pPS 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 (3, 4, 5, 6, 7, 8, 12, 16, 21, 22)
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2. A method for obtaining differentiated cells from a donor culture of undifferentiated primate pluripotent stem (pPS) cells, comprising:
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a) culturing the pPS cells on a solid surface in an environment essentially free of feeder cells;
b) changing medium used to culture the cells so that they differentiate before there is overgrowth or formation of colonies; and
c) harvesting differentiated cells from the solid surface. - View Dependent Claims (9, 10, 11, 13, 14, 15, 17, 18, 19, 20)
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Specification