Human embryonic stem cells genetically modified to contain a nucleic acid and cultured with fibroblast growth factor
This disclosure provides an improved system for culturing human pluripotent stem cells. Traditionally, pluripotent stem cells are cultured on a layer of feeder cells (such as mouse embryonic fibroblasts) to prevent them from differentiating. In the system described here, the role of feeder cells is replaced by components added to the culture environment that support rapid proliferation without differentiation. Effective features are a suitable support structure for the cells, and an effective medium that can be added fresh to the culture without being preconditioned by another cell type. Culturing human embryonic stem cells in fresh medium according to this invention causes the cells to expand surprisingly rapidly, while retaining the ability to differentiate into cells representing all three embryonic germ layers. This new culture system allows for bulk proliferation of pPS cells for commercial production of important products for use in drug screening and human therapy.
1. A method of genetically modifying human embryonic stem (“hES”) cells, comprising:
a. culturing hES cells essentially feeder free on an extracellular matrix in a non-conditioned nutrient media comprising fibroblast growth factor (FGF); and
b. introducing a nucleic acid into the hES cells of a) thereby genetically modifying the hES cells.
2. The method of claim 1 , wherein the extracellular matrix comprises at least one of fibronectin, laminin, proteoglycan, entactin, and heparin sulfate.
3. The method of claim 1 , wherein the extracellular matrix comprises collagen.
4. The method of claim 1 , further comprising preferentially selecting a genetically modified hES cell.
5. The method of claim 1 , wherein the nucleic acid is a vector or a plasmid.
6. The method of claim 5 , wherein the vector is a viral vector.
7. The method of claim 1 , wherein the introduced nucleic acid is present in a lipid/DNA complex.
8. The method of claim 1 , wherein the nucleic acid comprises a promoter.
9. The method of claim 8 , wherein the promoter is chosen from the OCT-4 promoter and the hTERT promoter.
10. The method of claim 1 , wherein the nucleic acid comprises a drug sensitivity gene or a drug resistance gene.
11. An essentially feeder free cell culture comprising: a) a genetically modified human embryonic stem (“hES”) cell; b) an extra cellular matrix; and c) a non-conditioned nutrient media comprising fibroblast growth factor (FGF).
12. The cell culture of claim 11 , wherein the extracellular matrix comprises at least one of fibronectin, laminin, proteoglycan, entactin, and heparin sulfate.
13. The cell culture of claim 11 , wherein the extracellular matrix comprises collagen.